Sirna compounds comprising terminal substitutions

ABSTRACT

The invention relates to modified siRNA compounds which down-regulate target gene expression, to pharmaceutical compositions comprising such compounds and to methods of treating and/or preventing the incidence or severity of various diseases or conditions associated with the genes and/or symptoms associated with such diseases or conditions.

RELATED APPLICATIONS

This application is a §371 national stage of PCT InternationalApplication No. PCT/US2010/058123, filed Nov. 25, 2010, claiming thebenefit of U.S. Provisional Application Nos. 61/264,668, filed Nov. 26,2009; 61/295,721, filed Jan. 17, 2010; and 61/372,072, filed Aug. 9,2010, the contents of each of which are hereby incorporated by referencein their entirety.

REFERENCE TO SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“120521_(—)2094_(—)84023_Substitute_Sequence_Listing_GC.txt,” which is13.1 kilobytes in size, and which was created May 14, 2012 in the IBM-PCmachine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed May 14, 2012 aspart of this application.

FIELD OF THE INVENTION

The present invention relates to modified siRNA compounds,pharmaceutical compositions comprising same and methods of use thereoffor the inhibition of gene expression. The compounds and compositionsexhibit beneficial properties including knock down activity of targetgenes and are useful in the treatment of subjects suffering fromdiseases or conditions and or symptoms associated with such diseases orconditions or at risk of contracting diseases or conditions in whichgene expression has adverse consequences.

BACKGROUND OF THE INVENTION

PCT Patent Publication Nos. WO 2008/104978 and WO 2009/044392 to theassignee of the present invention and hereby incorporated by referencein their entirety, disclose novel siRNA structures.

There remains a need for active and effective siRNA therapeutic agentswhich exhibit enhanced knock down activity, increased stability and orreduced off target effects.

SUMMARY OF THE INVENTION

The double stranded RNA (dsRNA) compounds disclosed herein possessstructures and modifications which may, for example increase activity,increase stability, reduce immunogenicity, enhance loading into the RISCcomplex, and or minimize toxicity; the novel modifications of the siRNAsare beneficially applied to double stranded RNA useful in preventing orattenuating target gene expression.

Provided herein are double stranded (duplex) oligonucleotide compoundsuseful for the down regulation of gene expression. The presentdisclosure is based in part on the unexpected observation that a doublestranded nucleic acid molecule comprising a sense strand and acomplementary antisense strand and having a mismatch between the 5′terminal nucleotide of the antisense strand and the nucleotide of thetarget RNA show potent activity in down regulating target genes. Inpreferred embodiments the dsRNA comprises an adenosine, deoxyadenosine,uridine, deoxyuridine, ribothymidine or thymidine substituted atposition 1 (5′ terminus) of the antisense strand rather in place of acytidine or guanine.

According to one aspect provided are modified double stranded nucleicacid molecules having structure (A) set forth below:

(A) 5′    N¹-(N)x-Z 3′ (antisense strand) 3′ Z′-N²-(N′)y-z″ 5′(sense strand)wherein each of N², N and N′ is an unmodified or modifiedribonucleotide, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;wherein each of x and y is independently an integer between 17 and 39;wherein the sequence of (N′)y has complementarity to the sequence of(N)x and (N)x has complementarity to a consecutive sequence in a targetRNA;wherein N¹ is covalently bound to (N)x and is mismatched to the targetRNA or is a complementary DNA moiety to the target RNA;wherein N¹ is a moiety selected from the group consisting of natural ormodified uridine, deoxyribouridine, ribothymidine, deoxyribothymidine,adenosine or deoxyadenosine;wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N²— (N′)y; andwherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides, consecutivenon-nucleotide moieties or a combination thereof covalently attached atthe 3′ terminus of the strand in which it is present.

In some embodiments the sequence of (N′)y is fully complementary to thesequence of (N)x. In various embodiments sequence of N²—(N′)y iscomplementary to the sequence of N¹—(N)x. In some embodiments (N)xcomprises an antisense that is fully complementary to about 17 to about39 consecutive nucleotides in a target RNA. In other embodiments (N)xcomprises an antisense that is substantially complementary to about 17to about 39 consecutive nucleotides in a target RNA.

In some embodiments N¹ and N² form a Watson-Crick base pair. In someembodiments N¹ and N² form a non-Watson-Crick base pair. In someembodiments a base pair is formed between a ribonucleotide and adeoxyribonucleotide.

In some embodiments x=y=18, x=y=19 or x=y=20. In preferred embodimentsx=y=18.

In some embodiments N¹ is covalently bound to (N)x and is mismatched tothe target RNA. In various embodiments N¹ is covalently bound to (N)xand is a DNA moiety complementary to the target RNA.

In some embodiments a uridine in position 1 of the antisense strand issubstituted with an N¹ selected from adenosine, deoxyadenosine,deoxyuridine, ribothymidine or deoxythymidine. In various embodiments N¹selected from adenosine, deoxyadenosine or deoxyuridine.

In some embodiments guanosine in position 1 of the antisense strand issubstituted with an N¹ selected from adenosine, deoxyadenosine, uridine,deoxyuridine, ribothymidine or deoxythymidine. In various embodiments N¹is selected from adenosine, deoxyadenosine, uridine or deoxyuridine.

In some embodiments cytidine in position 1 of the antisense strand issubstituted with an N¹ selected from adenosine, deoxyadenosine, uridine,deoxyuridine, ribothymidine or deoxythymidine. In various embodiments N¹is selected from adenosine, deoxyadenosine, uridine or deoxyuridine.

In some embodiments adenosine in position 1 of the antisense strand issubstituted with an N¹ selected from deoxyadenosine, deoxyuridine,ribothymidine or deoxythymidine. In various embodiments N¹ selected fromdeoxyadenosine or deoxyuridine.

In some embodiments N¹ and N² form a base pair between uridine ordeoxyuridine, and adenosine or deoxyadenosine. In other embodiments N¹and N² form a base pair between deoxyuridine and adenosine.

In some embodiments the double stranded nucleic acid molecule is asiRNA, siNA or a miRNA.

The following table provides examples of N1 and corresponding N2.

5′ terminal nucleotide of AS Target with full match N1 (5′ terminal N2(3′ terminal nucleotide to target position of AS) position of SEN) A UrA, dA rU, dU, rT, dT A U dU, rT, dT rA, dA C G rA, dA rU, dU, rT, dT CG rU, dU, rT, dT rA, dA G C rA, dA rU, dU, rT, dT G C rU, dU, rT, dT rA,dA U A dA rU, dU rT, dT U A dU rT, dT rA, dA

In some embodiments of Structure (A), N¹ comprises uridine or adenosine.In certain embodiments of structure (A), N² comprises a 2′OMe sugarmodified ribonucleotide. In some embodiments of Structure (A), N¹comprises 2′OMe sugar-modified ribouridine and N² comprises adenosine ormodified adenosine. In some embodiments of Structure (A), N¹ comprisesadenosine and N² comprises a ribouridine or modified ribouridine.

In some embodiments Z and Z′ are absent. In other embodiments one of Zor Z′ is present.

In some embodiments each of N and N′ is an unmodified ribonucleotide. Insome embodiments at least one of N or N′ comprises a chemically modifiedribonucleotide or an unconventional moiety. In some embodiments theunconventional moiety is selected from a mirror nucleotide, an abasicribose moiety and an abasic deoxyribose moiety. In some embodiments theunconventional moiety is a mirror nucleotide, preferably an L-DNAmoiety. In some embodiments at least one of N or N′ comprises a 2′OMesugar-modified ribonucleotide.

In some embodiments the sequence of (N′)y is fully complementary to thesequence of (N)x. In other embodiments the sequence of (N′)y issubstantially complementary to the sequence of (N)x.

In some embodiments (N)x comprises an antisense sequence that is fullycomplementary to about 17 to about 39 consecutive nucleotides in atarget mRNA. In other embodiments (N)x comprises an antisense that issubstantially complementary to about 17 to about 39 consecutivenucleotides in a target mRNA. In some embodiments x=y=18.

According to certain preferred embodiments siRNA compounds comprisingone or more modified nucleotide, wherein the modified nucleotidepossesses a modification in the sugar moiety, in the base moiety or inthe internucleotide linkage moiety are provided.

Structure (A) motif is useful with any oligonucleotide pair (sense andantisense strands) to a mammalian or non-mammalian gene. In someembodiments the mammalian gene is a human gene.

In some embodiments a modified siRNA compound having structure (A)exhibits beneficial properties including enhanced activity (e.g. reducedIC50, increased knock down, reduced residual mRNA) when compared to acontrol compound, i.e. an siRNA compound wherein the antisenseoligonucleotide is fully complementary (including 5′ terminal nucleotidebase paired e.g. A-U, U-A, C-G, G-C) to a nucleotide sequence in atarget mRNA. In some embodiments the activity is enhanced by at least5%, by at least 10%, by at least 20%, by at least 25% or more whencompared to a control compound.

In another aspect the present invention provides a method of generatinga double stranded RNA molecule consisting of a sense strand and anantisense strand comprising the steps of

a) selecting a consecutive 17 to 25 nucleotide sequence in a target RNAand synthesizing an antisense strand comprising complementarity to theconsecutive 17 to 25 nucleotide sequence of the target mRNA wherein the5′ terminal nucleotide of the antisense strand is substituted withuridine, modified uridine, ribothymidine, deoxyribothymidine, adenosine,modified adenosine, deoxyadenosine or modified deoxyadenosine, with theproviso that a rG:rU wobble is not generated between the 5′ terminalnucleotide of the antisense strand and the 3′ terminal nucleotide of thetarget mRNA;b) synthesizing a sense strand of 17 to 25 nucleotides havingcomplementarity to the antisense strand, wherein the 3′ terminalnucleotide of the sense strand forms a Watson Crick base pair with the5′ terminal nucleotide of the guide strand; andc) annealing the antisense and sense strands; thereby generating adouble stranded RNA molecule.

According to one aspect, the present invention provides a method ofgenerating a a double stranded RNA molecule consisting of a sense strandand an antisense strand exhibiting enhanced RNAi activity when comparedto an unmodified a double stranded RNA molecule comprising the steps of

a) selecting a consecutive 17 to 25 nucleotide sequence in a target mRNAand synthesizing a sense strand comprising the consecutive 17 to 25nucleotide sequence of the target mRNA wherein the 3′ terminalnucleotide is substituted with adenosine, modified adenosine,deoxyadenosine or modified deoxyadenosine;b) synthesizing an antisense strand of 17 to 25 nucleotides havingcomplementarity to the sense strand wherein the 5′ terminal nucleotidecomprises ribouridine, modified ribouridine, deoxyribouridine ormodified deoxyribouridine and base pairs with the 3′ terminal nucleotideof the passenger strand;c) annealing the sense strand to the antisense strand;thereby generating a double stranded RNA molecule having enhanced RNAiactivity.

In some embodiments the modified double stranded RNA molecule exhibitsenhanced RNAi activity when compared to an unmodified siRNA duplex, i.e.a duplex having full match to the target mRNA.

According to another aspect, the present invention provides a method ofgenerating a modified a double stranded RNA molecule consisting of asense strand and antisense strand exhibiting enhanced RNAi activity whencompared to an unmodified a double stranded RNA molecule comprising thesteps of

a) selecting a consecutive 17 to 25 nucleotide sequence in a target mRNAand synthesizing a sense strand comprising the consecutive 17 to 25nucleotide sequence of the target mRNA wherein the 3′ terminalnucleotide is substituted with adenosine, modified adenosine,deoxyadenosine or modified deoxyadenosine;b) synthesizing an antisense strand of 17 to 25 nucleotides havingcomplementarity to the sense strand wherein the 5′ terminal nucleotidecomprises ribouridine, modified ribouridine, deoxyribouridine ormodified deoxyribouridine and base pairs with the 3′ terminal nucleotideof the sense strand;c) annealing the sense strand to the antisense strand;thereby generating a double stranded RNA molecule having enhanced RNAiactivity.

In some embodiments step a) includes selecting a consecutive 17 to 25nucleotide, or 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide sequencein a target RNA in a target cell wherein the 3′ terminal nucleotide isother than adenosine.

In another aspect provided is a pharmaceutical composition comprising acompound according to Structure (A), in an amount effective to inhibitmammalian or non-mammalian gene expression; and a pharmaceuticallyacceptable carrier. In some embodiments the mammalian gene is a humangene. In some embodiments the non-mammalian gene is involved in amammalian disease, preferably human disease.

Further provided are methods for treating or preventing the incidence orseverity of a disease or condition and/or for reducing the risk orseverity of a disease or condition in a subject in need thereof whereinthe disease or condition and/or a symptom and/or risk associatedtherewith is associated with expression of a mammalian or anon-mammalian gene. In a preferred embodiment the subject is a humansubject.

In some embodiments the disease or condition is selected from the grouphearing loss, acute renal failure (ARF), Delayed Graft Function (DGF)after kidney transplantation, glaucoma, ocular ischemic conditionsincluding anterior ischemic optic neuropathy, age-related maculardegeneration (AMD), Ischemic Optic Neuropathy (ION), dry eye syndrome,acute respiratory distress syndrome (ARDS) and other acute lung andrespiratory injuries, chronic obstructive pulmonary disease (COPD),primary graft failure, ischemia-reperfusion injury, reperfusion injury,reperfusion edema, allograft dysfunction, pulmonary reimplantationresponse and/or primary graft dysfunction (PGD) after organtransplantation, in particular in lung transplantation, organtransplantation including lung, liver, heart, pancreas, and kidneytransplantation, nephro- and neurotoxicity, spinal cord injury, braininjury, neurodegenerative disease or condition, pressure sores, oralmucositis fibrotic conditions including liver fibrosis, lung fibrosis;and cancer. Such methods involve administering to a mammal in need ofsuch treatment a prophylactically or therapeutically effective amount ofone or more such compounds, which inhibit or reduce expression oractivity of at least one such gene.

The compounds, methods, materials, and examples that will now bedescribed are illustrative only and are not intended to be limiting;materials and methods similar or equivalent to those described hereincan be used in practice or testing of the invention. Other features andadvantages of the invention will be apparent from the following detaileddescription, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of the dsRNA molecules as provided herein. In oneembodiment, the 5′ terminal G or C nucleotide of the antisense strand issubstituted with U, dU, rA, dA, rT, dT; or the 5′ terminal U nucleotideof the antisense strand is substituted with dU, rA, dA, rT, dT; or the5′ terminal A nucleotide of the antisense strand is substituted with U,dU, dA, rT, dT. In one embodiment the 5′ terminal nucleotide of theantisense strand is mismatched to the target mRNA. In another embodimentthe 5′ terminal nucleotide of the antisense strand is adeoxyribonucleotide complementary to the target mRNA.

FIGS. 2-10 show activity (% residual target remaining) bar graphs ofdsRNA molecules with different nucleotides in position 1 of theantisense strand to targets that have been modified to individuallyinclude the four ribonucleotides at position 19 (position 1 of AS).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to oligonucleotide compoundswhich down-regulate expression of various genes, particularly to smallinterfering RNAs (siRNA), specifically to modified siRNA compounds andto the use of these modified siRNA compounds in preparation ofpharmaceutical compositions and in treatment of a subject suffering fromvarious medical conditions.

The compounds and compositions exhibit beneficial properties includingpotent knock down activity of target genes and are useful in thetreatment of subjects suffering from diseases or conditions and orsymptoms associated with such diseases or conditions or at risk ofcontracting diseases or conditions in which gene expression has adverseconsequences.

Accordingly, in certain aspects modified siRNA compounds andpharmaceutical compositions comprising same useful in down regulatinggene expression are provided.

In another aspect, the present invention provides a method of treating asubject suffering from or susceptible to a microvascular disorder, eyedisease or disorder, hearing impairment (including hearing loss), arespiratory (including pulmonary) disorder, neurodegenerative disease ordisorder, spinal cord injury, brain injury angiogenesis- andapoptosis-related conditions comprising administering to the subject apharmaceutical composition comprising at least one small interfering RNAof the invention that targets a mammalian or non-mammalian gene, in anamount sufficient to down-regulate the expression of the gene.

In certain embodiments, the subject compounds are useful in inhibitingexpression of a target gene for treatment of inter alia, respiratorydisorders, microvascular disorders or eye disorders. Particular diseasesand conditions to be treated are ARDS; COPD; ALI; Emphysema; DiabeticNeuropathy, nephropathy and retinopathy; DME and other diabeticconditions; Glaucoma; AMD; BMT retinopathy; ischemic conditionsincluding stroke; OIS; neurodegenerative disorders such as Parkinson's,Alzheimer's, ALS; kidney disorders: ARF, DGF, transplant rejection;hearing disorders; spinal cord injuries; oral mucositis; cancer, dry eyesyndrome and pressure sores.

DEFINITIONS

For convenience certain terms employed in the specification, examplesand claims are described herein.

It is to be noted that, as used herein, the singular forms “a”, “an” and“the” include plural forms unless the content clearly dictatesotherwise. Where aspects or embodiments of the invention are describedin terms of Markush groups or other grouping of alternatives, thoseskilled in the art will recognize that the invention is also therebydescribed in terms of any individual member or subgroup of members ofthe group.

An “inhibitor” is a compound, which is capable of reducing (partially orfully) the expression of a gene or the activity of the product of suchgene to an extent sufficient to achieve a desired biological orphysiological effect. The term “inhibitor” as used herein refers to asiRNA inhibitor. A “siRNA inhibitor” is a compound that is capable ofreducing the expression of a gene or the activity of the product of suchgene to an extent sufficient to achieve a desired biological orphysiological effect. The term “siRNA inhibitor” as used herein refersto one or more of a siRNA, shRNA, siNA, synthetic shRNA; miRNA.Inhibition may also be referred to as down-regulation or, for RNAi,silencing.

The term “inhibit” as used herein refers to reducing the expression of agene or the activity of the product of such gene to an extent sufficientto achieve a desired biological or physiological effect Inhibition iseither complete or partial

As used herein, the term “inhibition” of a target gene means inhibitionof the gene expression (transcription or translation) or polypeptideactivity of the product of a target gene. “Gene product” as used herein,refers to a product of a gene such as an RNA transcript or apolypeptide. The terms “RNA transcript”, “mRNA polynucleotide sequence”,“mRNA sequence” and “mRNA” are used interchangeably.

As used herein, the terms “polynucleotide” and “nucleic acid” may beused interchangeably and refer to nucleotide sequences comprisingdeoxyribonucleic acid (DNA), and ribonucleic acid (RNA). The terms areto be understood to include, as equivalents, analogs of either RNA orDNA made from nucleotide analogs. Throughout this disclosure, mRNAsequences are set forth as representing the corresponding genes.

“Oligonucleotide” or “oligomer” refers to a deoxyribonucleotide orribonucleotide sequence from about 2 to about 50 nucleotides. Each DNAor RNA nucleotide may be independently natural or synthetic, and ormodified or unmodified. Modifications include changes to the sugarmoiety, the base moiety and or the linkages between nucleotides in theoligonucleotide. The compounds of the present invention encompassmolecules comprising deoxyribonucleotides, ribonucleotides, modifieddeoxyribonucleotides, modified ribonucleotides, nucleotide analogues,modified nucleotide analogues, unconventional and abasic moieties andcombinations thereof.

Substantially complementary refers to complementarity of greater thanabout 84%, to another sequence. For example in a duplex regionconsisting of 19 base pairs one mismatch results in 94.7%complementarity, two mismatches results in about 89.5% complementarityand 3 mismatches results in about 84.2% complementarity, rendering theduplex region substantially complementary. Accordingly substantiallyidentical refers to identity of greater than about 84%, to anothersequence.

“Nucleotide” is meant to encompass deoxyribonucleotides andribonucleotides, which may be natural or synthetic and modified orunmodified. Nucleotides include known nucleotide analogues, which aresynthetic, naturally occurring, and non-naturally occurring. Examples ofsuch analogs include, without limitation, phosphorothioates,phosphoramidites, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).Modifications include changes to the sugar moiety, the base moiety andor the linkages between ribonucleotides in the oligoribonucleotide. Asused herein, the term “ribonucleotide” encompasses natural andsynthetic, unmodified and modified ribonucleotides and ribonucleotideanalogues which are synthetic, naturally occurring, and non-naturallyoccurring. Modifications include changes to the sugar moiety, to thebase moiety and/or to the linkages between ribonucleotides in theoligonucleotide.

The nucleotides are selected from naturally occurring or syntheticmodified bases. Naturally occurring bases include adenine, guanine,cytosine, thymine and uracil.

Modified bases of nucleotides include inosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines,5-halouracil, 5-halocytosine, 6-azacytosine and 6-az thymine,pseudouracil, deoxypseudouracil, 4-thiouracil, ribo-2-thiouridine,ribo-4-thiouridine, 8-haloadenine, 8-aminoadenine, 8-thioladenine,8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substitutedadenines, 8-haloguanines, 8-aminoguanine, 8-thiolguanine,8-thioalkylguanines 8-hydroxylguanine and other substituted guanines,other aza and deaza adenines, other aza and deaza guanines,5-methylribouridine, 5-trifluoromethyl uracil, 5-methylribocytosine, and5-trifluorocytosine. In some embodiments one or more nucleotides in anoligomer is substituted with inosine.

In some embodiments the siRNA compound further comprises at least onemodified ribonucleotide selected from the group consisting of aribonucleotide having a sugar modification, a base modification or aninternucleotide linkage modification and may contain DNA, and modifiednucleotides such as LNA (locked nucleic acid), ENA (ethylene-bridgednucleic acid), PNA (peptide nucleic acid), arabinoside,phosphonocarboxylate or phosphinocarboxylate nucleotide (PACEnucleotide), or nucleotides with a 6 carbon sugar.

Modified deoxyribonucleotide includes, for example 5′OMe DNA(5-methyl-deoxyriboguanosine-3′-phosphate) which may be useful as anucleotide in the 5′ terminal position (position number 1); PACE(deoxyriboadenosine 3′ phosphonoacetate, deoxyribocytidine 3′phosphonoacetate, deoxyriboguanosine 3′ phosphonoacetate,deoxyribothymidine 3′ phosphonoacetate).

Bridged nucleic acids include LNA (2′-O, 4′-C-methylene bridged NucleicAcid adenosine 3′ monophosphate, 2′-O,4′-C-methylene bridged NucleicAcid 5-methyl-cytidine 3′ monophosphate, 2′-O,4′-C-methylene bridgedNucleic Acid guanosine 3′ monophosphate, 5-methyl-uridine (or thymidine)3′ monophosphate); and ENA (2′-O,4′-C-ethylene bridged Nucleic Acidadenosine 3′ monophosphate, 2′-O,4′-C-ethylene bridged Nucleic Acid5-methyl-cytidine 3′ monophosphate, 2′-O,4′-C-ethylene bridged NucleicAcid guanosine 3′ monophosphate, 5-methyl-uridine (or thymidine) 3′monophosphate).

All analogs of, or modifications to, a nucleotide/oligonucleotide areemployed with the present invention, provided that said analog ormodification does not substantially adversely affect the properties,e.g. function, of the nucleotide/oligonucleotide.

Acceptable modifications include modifications of the sugar moiety,modifications of the base moiety, modifications in the internucleotidelinkages and combinations thereof.

A sugar modification includes a modification on the 2′ moiety of thesugar residue and encompasses amino, fluoro, alkoxy (e.g. methoxy),alkyl, amino, fluoro, chloro, bromo, CN, CF, imidazole, carboxylate,thioate, C1 to C10 lower alkyl, substituted lower alkyl, alkaryl oraralkyl, OCF₃, OCN, O—, S—, or N— alkyl; O—, S—, or N-alkenyl; SOCH₃;SO₂CH₃; ONO₂; NO₂, N₃; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino or substituted silyl, as, among others,described in European patents EP 0 586 520 B1 or EP 0 618 925 B1.

In one embodiment the modified siRNA compound comprises at least oneribonucleotide comprising a 2′ modification on the sugar moiety (“2′sugar modification”). In certain embodiments the siRNA compoundcomprises 2′O-alkyl or 2′-fluoro or 2′O-allyl or any other 2′modification, optionally on alternate positions. Other stabilizingmodifications are also possible (e.g. terminal modifications). In someembodiments a preferred 2′O-alkyl is 2′O-methyl (methoxy) sugarmodification.

In some embodiments the backbone of the oligonucleotides is modified andcomprises phosphate-D-ribose entities but may also containthiophosphate-D-ribose entities, triester, thioate, 2′-5′ bridgedbackbone (also may be referred to as 5′-2′), PACE and the like.

As used herein, the terms “non-pairing nucleotide analog” means anucleotide analog which comprises a non-base pairing moiety includingbut not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole,3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me riboU, N3-Me riboT, N3-MedC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In someembodiments the non-base pairing nucleotide analog is a ribonucleotide.In other embodiments the non-base pairing nucleotide analog is adeoxyribonucleotide. In addition, analogues of polynucleotides may beprepared wherein the structure of one or more nucleotide isfundamentally altered and better suited as therapeutic or experimentalreagents. An example of a nucleotide analogue is a peptide nucleic acid(PNA) wherein the deoxyribose (or ribose) phosphate backbone in DNA (orRNA) is replaced with a polyamide backbone which is similar to thatfound in peptides. PNA analogues have been shown to be resistant toenzymatic degradation and to have enhanced stability in vivo and invitro. Other modifications include polymer backbones, cyclic backbones,acyclic backbones, thiophosphate-D-ribose backbones, triester backbones,thioate backbones, 2′-5′ bridged backbone, artificial nucleic acids,morpholino nucleic acids, glycol nucleic acid (GNA), threose nucleicacid (TNA), arabinoside, and mirror nucleoside (for example,beta-L-deoxyribonucleoside instead of beta-D-deoxyribonucleoside).Examples of siRNA compounds comprising LNA nucleotides are disclosed inElmen et al., (NAR 2005, 33(1):439-447).

In some embodiments the compounds of the present invention aresynthesized with one or more inverted nucleotides, for example invertedthymidine or inverted adenosine (see, for example, Takei, et al., 2002,JBC 277(26):23800-06).

Other modifications include 3′ terminal modifications also known ascapping moieties. Such terminal modifications are selected from anucleotide, a modified nucleotide, a lipid, a peptide, a sugar andinverted abasic moiety. Such modifications are incorporated, for exampleat the 3′ terminus of the sense and/or antisense strands.

What is sometimes referred to in the present invention as an “abasicnucleotide” or “abasic nucleotide analog” is more properly referred toas a pseudo-nucleotide or an unconventional moiety. A nucleotide is amonomeric unit of nucleic acid, consisting of a ribose or deoxyribosesugar, a phosphate, and a base (adenine, guanine, thymine, or cytosinein DNA; adenine, guanine, uracil, or cytosine in RNA). A modifiednucleotide comprises a modification in one or more of the sugar,phosphate and or base. The abasic pseudo-nucleotide lacks a base, andthus is not strictly a nucleotide.

The term “capping moiety” as used herein includes abasic ribose moiety,abasic deoxyribose moiety, modifications abasic ribose and abasicdeoxyribose moieties including 2′ O alkyl modifications; inverted abasicribose and abasic deoxyribose moieties and modifications thereof;C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5′O-Menucleotide; and nucleotide analogs including 4′,5′-methylene nucleotide;1-(β-D-erythrofuranosyl)nucleotide; 4′-thionucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate;5′-amino; and bridging or non bridging methylphosphonate and 5′-mercaptomoieties.

Certain preferred capping moieties are abasic ribose or abasicdeoxyribose moieties; inverted abasic ribose or abasic deoxyribosemoieties; C6-amino-Pi; a mirror nucleotide including L-DNA and L-RNA.

A “hydrocarbon moiety or derivative thereof” refers to straight chain orbranched alkyl moieties and moieties per se or further comprising afunctional group including alcohols, phosphodiester, phosphorothioate,phosphonoacetate and also includes amines, carboxylic acids, esters,amides aldehydes. “Hydrocarbon moiety” and “alkyl moiety” are usedinterchangeably.

“Terminal functional group” includes halogen, alcohol, amine,carboxylic, ester, amide, aldehyde, ketone, ether groups.

The term “unconventional moiety” as used herein refers to abasic ribosemoiety, an abasic deoxyribose moiety, a deoxyribonucleotide, a modifieddeoxyribonucleotide, a mirror nucleotide, a non-base pairing nucleotideanalog and a nucleotide joined to an adjacent nucleotide by a 2′-5′internucleotide phosphate bond; bridged nucleic acids including lockednucleic acids (LNA) and ethylene bridged nucleic acids (ENA).

Abasic deoxyribose moiety includes for example abasicdeoxyribose-3′-phosphate; 1,2-dideoxy-D-ribofuranose-3-phosphate;1,4-anhydro-2-deoxy-D-ribitol-3-phosphate.

Inverted abasic deoxyribose moiety includes inverted deoxyriboabasic;3′,5′ inverted deoxyabasic 5′-phosphate.

A “mirror” nucleotide is a nucleotide with reversed chirality to thenaturally occurring or commonly employed nucleotide, i.e., a mirrorimage (L-nucleotide) of the naturally occurring (D-nucleotide), alsoreferred to as L-RNA in the case of a mirror ribonucleotide, and“spiegelmer”. The nucleotide can be a ribonucleotide or adeoxyribonucleotide and my further comprise at least one sugar, base andor backbone modification. See U.S. Pat. No. 6,586,238. Also, U.S. Pat.No. 6,602,858 discloses nucleic acid catalysts comprising at least oneL-nucleotide substitution. Mirror nucleotide includes for example L-DNA(L-deoxyriboadenosine-3′-phosphate (mirror dA);L-deoxyribocytidine-3′-phosphate (mirror dC);L-deoxyriboguanosine-3′-phosphate (mirror dG);L-deoxyribothymidine-3′-phosphate (mirror dT) and L-RNA(L-riboadenosine-3′-phosphate (mirror rA); L-ribocytidine-3′-phosphate(mirror rC); L-riboguanosine-3′-phosphate (mirror rG);L-ribouridine-3′-phosphate (mirror dU).

According to one aspect the present invention provides inhibitorymodified siRNA compounds comprising unmodified ribonucleotides, modifiedribonucleotides and/or unconventional moieties. In some embodiments themodified siRNA compound comprises at least one modified nucleotideselected from the group consisting of a sugar modification, a basemodification and an internucleotide linkage modification and may containmodified nucleotides such as LNA (locked nucleic acid) including ENA(ethylene-bridged nucleic acid; PNA (peptide nucleic acid); arabinoside;PACE (phosphonoacetate and derivatives thereof), or nucleotides with asix-carbon sugar or an unconventional moiety selected from an abasicribose moiety, an abasic deoxyribose moiety, a modified or unmodifieddeoxyribonucleotide, a mirror nucleotide, and a nucleotide joined to anadjacent nucleotide by a 2′-5′ internucleotide phosphate bond. In someembodiments a modified ribonucleotide is a 2′OMe sugar modifiedribonucleotide.

In some embodiments the siRNA is blunt ended at the 3′ terminus of thecompound, i.e. the siRNA is blunt ended on the end defined by the3′-terminus of the sense or passenger strand and the 5′-terminus ofantisense or guide strand.

In other embodiments at least one of the two strands has a 3′ overhangof at least one nucleotide at the 3′-terminus; the overhang comprises atleast one deoxyribonucleotide. At least one of the strands optionallycomprises an overhang of at least one nucleotide at the 3′-terminus. Theoverhang consists of from about 1 to about 5 nucleotides.

In various embodiments the overhangs are independently selected from anucleotide, a non-nucleotide and a combination thereof. In certainembodiments, each overhang, if present, is independently selected from aribonucleotide, deoxyribonucleotide, abasic deoxyribose moiety, abasicdeoxyribose moiety, C3-amino-Pi, C4-amino-Pi, C5-amino-Pi, C6-amino-Pi,a mirror nucleotide.

In some embodiments each of Z and/or Z′ independently includes a C2, C3,C4, C5 or C6 alkyl moiety, optionally a C3 [propane, —(CH2)₃-] moiety ora derivative thereof including propanol (C3-OH), propanediol, andphosphodiester derivative of propanediol (“C3Pi”). In preferredembodiments each of Z and/or Z′ includes two hydrocarbon moieties and insome examples is C3Pi-C3OH or C3Pi-C3Pi. Each C3 is covalentlyconjugated to an adjacent C3 via a covalent bond, preferably aphospho-based bond. In some embodiments the phospho-based bond is aphosphorothioate, a phosphonoacetate or a phosphodiester bond.

In a specific embodiment x=y=19 and Z comprises C3-C3. In someembodiments the C3-C3 overhang is covalently attached to the 3′ terminusof (N)x or (N′)y via a covalent linkage, for example a phosphodiesterlinkage. In some embodiments the linkage between a first C3 and a secondC3 is a phosphodiester linkage. In some embodiments the 3′non-nucleotide overhang is C3Pi-C3Pi. In some embodiments the 3′non-nucleotide overhang is C3Pi-C3Ps. In some embodiments the 3′non-nucleotide overhang is C3Pi-C3OH(OH is hydroxy). In some embodimentsthe 3′ non-nucleotide overhang is C3Pi-C3OH.

In various embodiments the alkyl moiety comprises an alkyl derivativeincluding a C3 alkyl, C4 alkyl, C5 alky or C6 alkyl moiety comprising aterminal hydroxyl, a terminal amino, or terminal phosphate group. Insome embodiments the alkyl moiety is a C3 alkyl or C3 alkyl derivativemoiety. In some embodiments the C3 alkyl moiety comprises propanol,propylphosphate, propylphosphorothioate or a combination thereof.

The C3 alkyl moiety is covalently linked to the 3′ terminus of (N′)yand/or the 3′ terminus of (N)x via a phosphodiester bond. In someembodiments the alkyl moiety comprises propanol, propyl phosphate orpropyl phosphorothioate.

In some embodiments each of Z and Z′ is independently selected frompropanol, propyl phosphate propyl phosphorothioate, combinations thereofor multiples thereof in particular 2 or 3 covalently linked propanol,propyl phosphate, propyl phosphorothioate or combinations thereof.

In some embodiments each of Z and Z′ is independently selected frompropyl phosphate, propyl phosphorothioate, propyl phospho-propanol;propyl phospho-propyl phosphorothioate; propylphospho-propyl phosphate;(propyl phosphate)₃, (propyl phosphate)₂-propanol, (propylphosphate)₂-propyl phosphorothioate. Any propane or propanol conjugatedmoiety can be included in Z or Z′.

The structures of exemplary 3′ terminal non-nucleotide moieties are asfollows:

In additional embodiments each of Z and/or Z′ comprises a combination ofan abasic moiety and an unmodified deoxyribonucleotide or ribonucleotideor a combination of a hydrocarbon moiety and an unmodifieddeoxyribonucleotide or ribonucleotide or a combination of an abasicmoiety (deoxyribo or ribo) and a hydrocarbon moiety. In suchembodiments, each of Z and/or Z′ comprises C3Pi-rAb or C3Pi-dAb.

The length of RNA duplex is from about 18 to about 40 ribonucleotides,preferably 19 to 23 ribonucleotides. In some embodiments the length ofeach strand (oligomer) is independently selected from the groupconsisting of about 18 to about 40 bases, preferably 18 to 23 bases andmore preferably 19, 20 or 21 ribonucleotides.

In some embodiments, the complementarity between the antisense strand ofthe modified siRNA compound and the target nucleic acid is perfect. Inother embodiments, the antisense strand of the modified siRNA compoundand the target nucleic acid are substantially complementary, i.e. havingone, two or up to three mismatches between said antisense strand and thetarget nucleic acid.

In certain embodiments the complementarity between the antisense strandand the sense strand of the modified siRNA compound of present inventionis perfect. In some embodiments, the strands are substantiallycomplementary, i.e. having one, two or up to three mismatches betweensaid antisense strand and said sense strand.

The siRNAs compounds of the present invention possess a terminal moietycovalently bound at the 5′-terminus of the antisense strand which ismismatched to a nucleotide in a target mRNA. In various embodiments themoiety at the 5′-terminus of the antisense strand is selected form thegroup consisting of ribouridine, deoxyribouridine, modified ribouridine,modified deoxyribouridine, pseudouracil, deoxypseudouracil,deoxyribothymidine, modified deoxyribothymidine, ribocytosine, modifiedribocytosine, deoxyribocytosine, modified deoxyribocytosine,5-methylribocytosine, modified 5-methylribocytosine,5-methylribouridine, ribo-2-thiouridine, ribo-4-thiouridine, abasicribose moiety and abasic deoxyribose moiety.

In some embodiments the modified siRNA compounds of the inventionexhibit enhanced activity, when compared to an siRNA compound whereinthe antisense strand including the 5′-terminal nucleotide is fullycomplementary to a consecutive sequence in a target mRNA.

The siRNA structures of the present invention are beneficially appliedto double stranded RNA useful in inhibiting or attenuating mammalian andnon-mammalian gene expression.

siRNA Oligonucleotides

In one aspect the present invention provides a compound having structure(A) set forth below:

(A) 5′    N¹-(N)x-Z 3′ (antisense strand) 3′ Z′-N²-(N′)y-z″ 5′(sense strand)wherein each of N², N and N′ is an unmodified or modifiedribonucleotide, or an unconventional moiety;wherein each of (N)x and (N′)y is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;wherein each of x and y is independently an integer between 17 and 39;wherein the sequence of (N′)y has complementarity to the sequence of(N)x and (N)x has complementarity to a consecutive sequence in a targetRNA;wherein N¹ is covalently bound to (N)x and is mismatched to the targetRNA or is a DNA moiety complementary to the target RNA;wherein N¹ is a moiety selected from the group consisting of natural ormodified uridine, deoxyribouridine, ribothymidine, deoxyribothymidine,adenosine or deoxyadenosine;wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N²—(N′)y; andwherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides, consecutivenon-nucleotide moieties or a combination thereof covalently attached atthe 3′ terminus of the strand in which it is present.

In some embodiments N¹ is covalently bound to (N)x and is mismatched tothe target RNA.

In some embodiments N¹ is covalently bound to (N)x and is a DNA moietycomplementary to the target RNA.

In some embodiments N¹ and N² together form a Watson-Crick base pair. Inother embodiments N¹ and N² together form a non-Watson-Crick base pair.

In some embodiments x=y=18. In other embodiments x=y=19. In yet otherembodiments x=y=20. In preferred embodiments x=y=18.

In some embodiments N¹ is a modified ribocytosine or a modifiedribouridine. In certain embodiments of N¹ modified ribocytosine ormodified ribouridine is selected from the group comprising5-methylribocytosine, modified 5-methylribocytosine,5-methylribouridine, ribo-2-thiouridine and ribo-4-thiouridine.

In certain embodiments N¹ is selected from the group consisting ofribocytosine, modified ribocytosine, deoxyribocytosine, modifieddeoxyribocytosine, 5-methylribocytosine and modified5-methylribocytosine. In other embodiments N¹ is selected from the groupconsisting of ribouridine, deoxyribouridine, modified ribouridine,modified deoxyribouridine, pseudouracil, deoxypseudouracil,5-methylribouridine, ribo-2-thiouridine and ribo 4-thiouridine. In someembodiments N¹ is deoxyribothymidine or modified deoxyribothymidine.

In some embodiments N² is a modified ribonucleotide or an unmodifiedribonucleotide. In certain embodiments N² is selected from the groupcomprising riboguanine, modified riboguanine, deoxyguanine, modifieddeoxyguanine, ribouridine, modified ribouridine, deoxyribouridine,modified deoxyribouridine, adenosine, modified adenosine,deoxyadenosine, modified deoxyadenosine, ribocytosine, modifiedribocytosine, deoxyribocytosine and modified deoxyribocytosine.

In some embodiments N2 is selected from the group consisting ofriboguanine, modified riboguanine, deoxyguanine, ribouridine,deoxyribouridine, adenosine, deoxyadenosine, ribocytosine anddeoxyribocytosine. In other embodiments N2 is selected from the groupconsisting of ribouridine, modified ribouridine, deoxyribouridine,modified deoxyribouridine, adenosine, modified adenosine,deoxyadenosine, modified deoxyadenosine, ribocytosine, modifiedribocytosine, deoxyribocytosine and modified deoxyribocytosine. In yetother embodiments N2 is selected from the group consisting ofribouridine, modified ribouridine, deoxyribouridine, modifieddeoxyribouridine, adenosine, modified adenosine, deoxyadenosine,modified deoxyadenosine, ribocytosine, modified ribocytosine,deoxyribocytosine and modified deoxyribocytosine.

In certain embodiments N¹ is selected from the group consisting ofribocytosine, modified ribocytosine, deoxyribocytosine, modifieddeoxyribocytosine, 5-methylribocytosine and modified5-methylribocytosine and N² is selected from the group consisting ofriboguanine, modified riboguanine, deoxyguanine, ribouridine,deoxyribouridine, adenosine, deoxyadenosine, ribocytosine anddeoxyribocytosine.

In some embodiments N¹ is selected from the group consisting ofribouridine, deoxyribouridine, modified ribouridine, modifieddeoxyribouridine, pseudouracil, deoxypseudouracil, 5-methylribouridine,ribo-2-thiouridine and ribo 4-thiouridine and N² is selected from thegroup consisting of ribouridine, modified ribouridine, deoxyribouridine,modified deoxyribouridine, adenosine, modified adenosine,deoxyadenosine, modified deoxyadenosine, ribocytosine, modifiedribocytosine, deoxyribocytosine and modified deoxyribocytosine.

In certain embodiments N¹ is deoxyribothymidine or modifieddeoxyribothymidine and N² is selected from the group consisting ofribouridine, modified ribouridine, deoxyribouridine, modifieddeoxyribouridine, adenosine, modified adenosine, deoxyadenosine,modified deoxyadenosine, ribocytosine, modified ribocytosine,deoxyribocytosine and modified deoxyribocytosine.

In some embodiments of Structure (A), N¹ comprises 2′OMe sugar modifiedribouridine or 2′OMe sugar modified ribocytosine. In certain embodimentsof structure (A), N² comprises a 2′OMe sugar modified ribonucleotide.

In some embodiments Z and Z′ are absent. In other embodiments one of Zor Z′ is present.

In various embodiments Z and Z′ are independently selected from anucleotide, a non-nucleotide and a combination thereof. In certainembodiments, each of Z and Z′, if present, is independently selectedfrom a ribonucleotide, deoxyribonucleotide, abasic deoxyribose moiety,abasic deoxyribose moiety, C3-amino-Pi, C4-amino-Pi, C5-amino-Pi,C6-amino-Pi, a mirror nucleotide. In some embodiments Z is present. Inother embodiments Z′ is present. In additional embodiments both Z and Z′are present. In some embodiments Z and Z′ are present and are identical.In further embodiments Z and Z′ are present and are different. In someembodiments Z and Z′ are independently 1, 2, 3, 4 or 5 non-nucleotidemoieties or a combination of 2, 3, 4, or 5 non-nucleotide moieties andnucleotides. In some embodiments each of Z and or Z′ comprises 2non-nucleotide moieties covalently linked to the 3′ terminus of thesiRNA strand via a phosphodiester bond. In some embodiments Z and Z′ arepresent and each one independently comprises one or more alkyl moietiesand or derivative thereof. In some embodiments, N² comprisesriboadenosine and N¹ comprises uridine (ribouridine).

A non-nucleotide moiety is selected from the group consisting of anabasic moiety, an inverted abasic moiety, an alkyl moiety or derivativethereof, and an inorganic phosphate. In some embodiments anon-nucleotide moiety is an alkyl moiety or derivative thereof. In someembodiments the alkyl moiety comprises a terminal functional groupincluding alcohol, a terminal amine, a terminal phosphate or a terminalphosphorothioate moiety.

In some embodiments Z is present and comprises one or morenon-nucleotide moieties selected from the group consisting of an abasicmoiety, an inverted abasic moiety, hydrocarbon moiety or derivativethereof, and an inorganic phosphate. In some embodiments Z is presentand comprises one or more alkyl moieties and or derivative thereof.

In additional embodiments Z′ is present and comprises one or morenon-nucleotide moieties selected from the group consisting of an abasicmoiety, an inverted abasic moiety, a hydrocarbon moiety, and aninorganic phosphate. In some embodiments Z′ is present and comprises oneor more alkyl moieties and or derivative thereof.

In additional embodiments x=y=18 and either Z or Z′ is present andindependently comprises two non-nucleotide moieties.

In additional embodiments x=y=18 and Z and Z′ are present and eachindependently comprises two non-nucleotide moieties.

In some embodiments each of Z and Z′ includes an abasic moiety, forexample a deoxyriboabasic moiety (referred to herein as “dAb”) orriboabasic moiety (referred to herein as “rAb”). In some embodimentseach of Z and/or Z′ comprises two covalently linked abasic moieties andis for example dAb-dAb or rAb-rAb or dAb-rAb or rAb-dAb. Each moiety iscovalently conjugated an adjacent moiety via a covalent bond, preferablya phospho-based bond. In some embodiments the phospho-based bond is aphosphorothioate, a phosphonoacetate or a phosphodiester bond.

In some embodiments each of Z and/or Z′ independently includes an alkylmoiety, optionally propane [(CH2)₃] moiety or a derivative thereofincluding propanol (C3-OH) and phospho derivative of propanediol(“C3-3′Pi”). In some embodiments each of Z and/or Z′ includes twohydrocarbon moieties and in some examples is C3-C3. Each C3 iscovalently conjugated an adjacent C3 via a covalent bond, preferably aphospho-based bond. In some embodiments the phospho-based bond is aphosphorothioate, a phosphonoacetate or a phosphodiester bond.

In a specific embodiment x=y=18 and Z comprises C3Pi-C3OH or C3Pi-C3Pi.In a specific embodiment x=y=18 and Z comprises C3Pi-C3OH or C3Pi-C3Pi.In some embodiments the C3-C3 overhang is covalently attached to the 3′terminus of (N)x or (N′)y via covalent linkage, for example aphosphodiester linkage. In some embodiments the linkage between a firstC3 and a second C3 is a phosphodiester linkage.

In various embodiments the alkyl moiety is a C3 alkyl to C6 alkyl moietycomprising a terminal hydroxyl, a terminal amino, terminal phosphategroup. In some embodiments the alkyl moiety is a C3 alkyl moiety. Insome embodiments the C3 alkyl moiety comprises propanol,propylphosphate, propylphosphorothioate or a combination thereof.

The C3 alkyl moiety is covalently linked to the 3′ terminus of (N′)y andor the 3′ terminus of (N)x via a phosphodiester bond. In someembodiments the alkyl moiety comprises propanol, propyl phosphate orpropyl phosphorothioate.

In some embodiments each of Z and Z′ is independently selected frompropanol, propyl phosphate, propyl phosphorothioate, combinationsthereof or multiples thereof.

In some embodiments each of Z and Z′ is independently selected frompropyl phosphate, propyl phosphorothioate, propyl phospho-propanol;propyl phospho-propyl phosphorothioate; propylphospho-propyl phosphate;(propyl phosphate)₃, (propyl phosphate)-2-propanol, (propylphosphate)₂-propyl phosphorothioate. Any propane or propanol conjugatedmoiety can be included in Z or Z′.

In additional embodiments each of Z and/or Z′ comprises a combination ofan abasic moiety and an unmodified deoxyribonucleotide or ribonucleotideor a combination of a hydrocarbon moiety and an unmodifieddeoxyribonucleotide or ribonucleotide or a combination of an abasicmoiety (deoxyribo or ribo) and a hydrocarbon moiety. In suchembodiments, each of Z and/or Z′ comprises C3-rAb or C3-dAb.

In preferred embodiments x=y=18, Z′ is absent, Z is present andcomprises two alkyl moieties covalently linked to each other via aphosphodiester bond, N² comprises riboadenosine and N¹ comprisesuridine.

In some embodiments N and N′ comprise an unmodified nucleotide. In someembodiments at least one of N or N′ comprises a chemically modifiedribonucleotide or an unconventional moiety. In some embodiments theunconventional moiety is selected from the group consisting of a mirrornucleotide, an abasic ribose moiety, an abasic deoxyribose moiety, adeoxyribonucleotide, a modified deoxyribonucleotide, a mirrornucleotide, a non-base pairing nucleotide analog, a bridged nucleic acidand a nucleotide joined to an adjacent nucleotide by a 2′-5′internucleotide phosphate bond. In some embodiments the unconventionalmoiety is a mirror nucleotide, preferably an L-DNA moiety. In someembodiments at least one of N or N′ is modified at one or more of thesugar, the base or linker. In certain embodiments at least one of N orN′ comprises a 2′OMe sugar modified ribonucleotide.

In certain embodiments (N)x and (N′)y are fully complementary. In otherembodiments (N)x and (N′)y are substantially complementary. In certainembodiments (N)x is fully complementary to a target sequence. In otherembodiments (N)x is substantially complementary to a target sequence.According to certain preferred embodiments the present inventionprovides a modified siRNA compound comprising one or more modifiednucleotide, wherein the modified nucleotide possesses a modification inthe sugar moiety, in the base moiety or in the internucleotide linkagemoiety.

In certain embodiments of the compound according to Structure (A)alternating ribonucleotides in each of (N)x and (N′)y are 2′-OMe sugarmodified ribonucleotides. In some embodiments of Structure (A) in (N)xthe nucleotides are unmodified or (N)x comprises alternating 2′OMe sugarmodified ribonucleotides and unmodified ribonucleotides; and theribonucleotide located at the middle position of N¹—(N)x being modifiedor unmodified preferably unmodified; wherein (N′)y comprises unmodifiedribonucleotides further comprising one modified nucleotide at a terminalor penultimate position.

In particular embodiments, x=y=18, N¹ comprises 2′OMe sugar modifiedribonucleotide, (N)x comprises 2′OMe sugar modified ribonucleotides andthe ribonucleotide located at the middle of N¹—(N)x is unmodified. Insome embodiments at least one nucleotide at either or both the 5′ and 3′termini of (N′)y are joined by a 2′-5′ phosphodiester bond. In someembodiments N¹ is joined to the 5′ terminus of (N)x by a 2′-5′phosphodiester bond. In some embodiments at least one nucleotides ateither or both the 5′ and 3′ termini of (N′)x are joined by a 2′-5′phosphodiester bond. In some embodiments N² is joined to the 3′ terminusof (N)y by a 2′-5′ phosphodiester bond. In certain embodiments x=y=18;in (N)x the nucleotides alternate between modified ribonucleotides andunmodified ribonucleotides, each modified ribonucleotide is a 2′OMesugar modified ribonucleotide and the ribonucleotide located at themiddle of N¹—(N)x being unmodified; N² is joined to the 3′ terminus of(N)y by a 2′-5′ phosphodiester bond and at least two nucleotides at the3′ terminus of (N′)y are joined by two 2′-5′ phosphodiester bonds. Inother preferred embodiments, x=y=18; in (N)x the nucleotides alternatebetween modified ribonucleotides and unmodified ribonucleotides, eachmodified ribonucleotide is a 2′-OMe sugar modified ribonucleotide andthe ribonucleotide located at the middle of N¹—(N)x being unmodified;and at least three consecutive nucleotides at the 5′ terminus of (N′)yare joined by three 2′-5′ phosphodiester bonds. In a further embodiment,the nucleotide located in the middle position of N²—(N)y, i.e.nucleotide at position 10, is a 2′OMe sugar modified ribonucleotide. Inanother preferred embodiment, in (N)x the nucleotides alternate between2′-OMe sugar modified ribonucleotides and unmodified ribonucleotides,and in (N′)y four consecutive nucleotides at the 5′ terminus are joinedby three 2′-5′ phosphodiester bonds and the 5′ terminal nucleotide ortwo or three consecutive nucleotides at the 5′ terminus comprise 3′-OMesugar modification.

In certain preferred embodiments, x=y=18 and in (N′)y the nucleotide inat least one position comprises a mirror nucleotide, adeoxyribonucleotide and a nucleotide joined to an adjacent nucleotide bya 2′-5′ internucleotide bond.

In certain embodiments, x=y=18 and (N′)y comprises a mirror nucleotide.In various embodiments the mirror nucleotide is an L-DNA nucleotide. Incertain embodiments the L-DNA is L-deoxyribocytidine. In someembodiments (N′)y comprises L-DNA at position 17. In other embodiments(N′)y comprises L-DNA at positions 16 and 17. In certain embodiments(N′)y comprises L-DNA nucleotides at positions 1 and at one or both ofpositions 16 and 17.

In yet other embodiments (N′)y comprises a DNA at position 14 and L-DNAat one or both of positions 16 and 17. In that structure, position 1 mayfurther comprise an L-DNA or an abasic unconventional moiety.

In other embodiments wherein x=y=20 the modifications for (N′)ydiscussed above instead of being on positions 14, 15, 16, 17 are onpositions 17, 18, 19, 20. For example, the modifications at one or bothof positions 16 and 17 are on one or both of positions 18 or 19 for the20-mer. All modifications in the 18-mer are similarly adjusted for the20- and 22-mer.

According to various embodiments in N²—(N′)y, N² is joined to the 3′terminus of (N)y by a 2′-5′ phosphodiester bond and in (N′)y 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 or 13 consecutive ribonucleotides at the 3′terminus are linked by 2′-5′ internucleotide linkages. In oneembodiment, N² is joined to the 3′ terminus of (N)y by a 2′-5′phosphodiester bond and three consecutive nucleotides at the 3′ terminusof (N′)y are joined by three 2′-5′ phosphodiester bonds, wherein one ormore of the 2′-5′ nucleotides which form the 2′-5′ phosphodiester bondsfurther comprises a 3′-OMe sugar modification. In some embodiments N²comprises a 2′-OMe sugar modification. In further embodiments the 3′terminal nucleotide of (N′)y comprises a 2′-OMe sugar modification. Incertain embodiments x=y=18 and in (N′)y two or more consecutivenucleotides at positions 14, 15, 16, 17 and 18 comprise a nucleotidejoined to an adjacent nucleotide by a 2′-5′ internucleotide bond. Invarious embodiments the nucleotide forming the 2′-5′ internucleotidebond comprises a 3′ deoxyribose nucleotide or a 3′ methoxy nucleotide.In some embodiments the nucleotides at positions 16 and 17 in (N′)y arejoined by a 2′-5′ internucleotide bond. In other embodiments thenucleotides at positions 15-16, 16-17, or 15-17 in (N′)y are joined by a2′-5′ internucleotide bond.

In certain embodiments (N′)y comprises an L-DNA at position 2 and 2′-5′internucleotide bonds at positions 15-16, 16-17, or 15-17.

In one embodiment the 3′ terminal nucleotide or two or three consecutivenucleotides at the 3′ terminus of (N′)y are L-deoxyribonucleotides.

In other embodiments in N²—(N′)y, N² is a 2′ sugar modified nucleotideand in (N′)y (N′)y 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13consecutive ribonucleotides at either terminus or 1 to 7 modifiednucleotides at each of the 5′ and 3′ termini are independently 2′ sugarmodified nucleotides. In some embodiments the 2′ sugar modificationcomprises the presence of an amino, a fluoro, an alkoxy or an alkylmoiety. In certain embodiments the 2′ sugar modification comprises amethoxy moiety (2′-OMe). In one series of embodiments, three, four orfive consecutive nucleotides at the 5′ terminus of (N′)y comprise the2′-OMe modification. In another embodiment, N² and two consecutivenucleotides at the 3′ terminus of (N′)y comprise the 2′-OMemodification.

In some embodiments in (N′)y 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13consecutive ribonucleotides at either or 1 to 7 modified nucleotides ateach of the 5′ and 3′ termini are independently bicyclic nucleotides. Invarious embodiments the bicyclic nucleotide is a locked nucleic acid(LNA) or a species of LNA, e.g. 2′-O, 4′-C-ethylene-bridged nucleic acid(ENA) is a species of LNA.

In various embodiments (N′)y comprises modified nucleotides at the 5′terminus or at both the 3′ and 5′ termini.

In some embodiments at least two nucleotides at the 5′ of (N′)y arejoined by P-ethoxy backbone modifications. In some embodiments N² at the3′ of (N′)y is joined by P-ethoxy backbone modifications. In certainembodiments x=y=18 and wherein in (N)x the nucleotides alternate betweenmodified ribonucleotides and unmodified ribonucleotides, each modifiedribonucleotide being a 2′OMe sugar modified ribonucleotide and theribonucleotide located at the middle position of N¹—(N)x beingunmodified; N1 being a 2′OMe sugar modified ribonucleotide and threeconsecutive nucleotides at the 3′ terminus are joined by two P-ethoxybackbone modifications or four consecutive nucleotides at the 5′terminus of (N′)y are joined by three P-ethoxy backbone modifications.In a further embodiment, N² at the 3′ of (N′)y is joined by P-ethoxybackbone modification.

In some embodiments in (N′)y 1, 2, 3, 4, 5, 6 or 7, consecutiveribonucleotides at each of the 5′ and 3′ termini are independentlymirror nucleotides, nucleotides joined by 2′-5′ phosphodiester bond, 2′sugar modified nucleotides or bicyclic nucleotide. In one embodiment,the modification at the 5′ and 3′ termini of (N′)y is identical. In oneembodiment, four consecutive nucleotides at the 5′ terminus of (N′)y arejoined by three 2′-5′ phosphodiester bonds and two or three consecutivenucleotides at the 3′ terminus of (N′)y are joined by two 2′-5′phosphodiester bonds. In a further embodiment N² at the 3′ of (N′)y isjoined at the 3′ terminus of (N′)y by a 2′-5′ phosphodiester bond. Inanother embodiment, the modification at the 5′ terminus of (N′)y isdifferent from the modification at the 3′ terminus of (N′)y. In onespecific embodiment, the modified nucleotides at the 5′ terminus of(N′)y are mirror nucleotides and the modified nucleotides at the 3′terminus of (N′)y are joined by 2′-5′ phosphodiester bond. In anotherspecific embodiment, three consecutive nucleotides at the 5′ terminus of(N′)y are LNA nucleotides and two consecutive nucleotides at the 3′terminus of (N′)y are joined by two 2′-5′ phosphodiester bonds. In (N)xthe nucleotides alternate between 2′OMe sugar modified ribonucleotidesand unmodified ribonucleotides, and the ribonucleotide located at themiddle of N¹—(N)x is unmodified.

In various embodiments, N² is joined at the 3′ terminus of (N′)y by a2′-5′ phosphodiester bond and two or three consecutive nucleotides atthe 3′ terminus of (N′)x are joined by one or two 2′-5′ phosphodiesterbonds. In some embodiments, N² is joined at the 3′ terminus of (N′)y bya 2′-5′ phosphodiester bond and two or three consecutive nucleotides atthe 3′ terminus of (N′)x are joined by one or two 2′-5′ phosphodiesterbonds and one or two or three consecutive nucleotides at the 3′ terminuscomprise 3′-OMe sugar modification. In a further embodiment N² comprise3′-OMe sugar modification.

In another embodiment the present invention provides a compound whereinx=y=18 and wherein in (N)x the nucleotides alternate between modifiedribonucleotides and unmodified ribonucleotides, each modifiedribonucleotide being a 2′OMe sugar modified ribonucleotide and theribonucleotide located at the middle of N¹—(N)x being unmodified; twonucleotides at the 3′ terminus of (N′)y are joined by two 2′-5′phosphodiester bonds and three nucleotides at the 5′ terminus of (N′)yare LNA such as ENA. In a further embodiment N² is joined at the 3′terminus of (N′)y by a 2′-5′ phosphodiester bond.

In another embodiment five consecutive nucleotides at the 5′ terminus of(N′)y comprise the 2′OMe sugar modification and two consecutivenucleotides at the 3′ terminus of (N′)y are L-DNA.

In yet another embodiment, the present invention provides a compoundwherein x=y=18 wherein (N)x consists of unmodified ribonucleotides; twoconsecutive nucleotides at the 3′ terminus of (N′)y are joined by a2′-5′ phosphodiester bonds and three consecutive nucleotides at the 5′terminus of (N′)y are LNA such as ENA. In a further embodiment N² isjoined at the 3′ terminus of (N′)y by a 2′-5′ phosphodiester bond.

According to other embodiments in (N′)y the 5′ or 3′ terminalnucleotide, or 2, 3, 4, 5 or 6 consecutive nucleotides at either terminior 1 to 4 nucleotides at each of the 5′ and 3′ termini are independentlyphosphonocarboxylate or phosphinocarboxylate nucleotides (PACEnucleotides). In some embodiments the PACE nucleotides aredeoxyribonucleotides. In some preferred embodiments in (N′)y, 1 or 2consecutive nucleotides at each of the 5′ and 3′ termini are PACEnucleotides.

In some embodiments, neither strand of the modified siRNA compounds ofthe invention is phosphorylated at the 3′ and 5′ termini. In otherembodiments the sense and antisense strands are phosphorylated at the 3′termini. In yet another embodiment, the antisense strand isphosphorylated at the terminal 5′ termini position using cleavable ornon-cleavable phosphate groups. In yet another embodiment, either orboth antisense and sense strands are phosphorylated at the 3′ terminiposition using cleavable or non-cleavable phosphate groups.

Structure (A) is useful with any oligonucleotide pair (sense andantisense strands) to a mammalian or non-mammalian gene. In someembodiments the mammalian gene is a human gene. Examples ofoligonucleotide sequence pairs are provided in PCT Patent PublicationNos. WO 2006/023544, WO 2007/084684, WO 2008/050329, WO 2007/141796, WO2009/044392, WO 2008/106102, WO 2008/152636, WO 2009/001359,WO/2009/090639 assigned to the assignee of the present invention andincorporated herein by reference in their entirety.

Unless otherwise indicated, in preferred embodiments of the structuresdiscussed herein the covalent bond between each consecutive N and N′ isa phosphodiester bond. Unless otherwise indicated, in preferredembodiments of the structures discussed herein the covalent bond betweenN¹ and (N)x and between N² and (N′)y is a phosphodiester bond. In someembodiments at least one of the covalent bond is a phosphorothioatebond.

For all of the structures above, in some embodiments the oligonucleotidesequence of (N)x is fully complementary to the oligonucleotide sequenceof (N′)y. In other embodiments the antisense and sense strands aresubstantially complementary. In certain embodiments (N)x is fullycomplementary to a mammalian mRNA or microbial RNA or viral RNA. Inother embodiments (N)x is substantially complementary to a mammalianmRNA or microbial RNA or viral RNA.

In some embodiments a modified siRNA compound having structure (A)exhibits beneficial properties including at least enhanced activity whencompared to an siRNA compound wherein N¹ is complementary to anucleotide in a target mRNA.

The present invention further provides a pharmaceutical compositioncomprising a compound of the present invention according to Structure(A), in an amount effective to inhibit mammalian or non-mammalian geneexpression, and a pharmaceutically acceptable carrier, and use thereoffor treatment of any one of the diseases and disorders disclosed herein.In some embodiments the mammalian gene is a human gene. In someembodiments the non-mammalian gene is involved in a mammalian disease,preferably human disease.

The present invention further relates to methods for treating orpreventing the incidence or severity of any one of the diseases orconditions disclosed herein or for reducing the risk or severity of adisease or a condition disclosed herein in a subject in need thereof,wherein the disease or condition and/or a symptom or risk associatedtherewith is associated with expression of a mammalian or anon-mammalian gene. In a preferred embodiment the subject is a humansubject.

siRNA Synthesis

Using public and proprietary algorithms the sense and antisensesequences of potential siRNAs are generated and N¹ and/or N² of thesiRNAs are substituted to generate the modified siRNA compoundsaccording to Structure (A).

siRNA molecules according to the above specifications are preparedessentially as described herein. The modified siRNA compounds of thepresent invention are synthesized by any of the methods that are wellknown in the art for synthesis of ribonucleic (or deoxyribonucleic)oligonucleotides. Synthesis is commonly performed in a commerciallyavailable synthesizer (available, inter alia, from Applied Biosystems).Oligonucleotide synthesis is described for example in Beaucage and Iyer,Tetrahedron 1992; 48:2223-2311; Beaucage and Iyer, Tetrahedron 1993; 49:6123-6194 and Caruthers, et. al., Methods Enzymol. 1987; 154: 287-313;the synthesis of thioates is, among others, described in Eckstein, Ann.Rev. Biochem. 1985; 54: 367-402, the synthesis of RNA molecules isdescribed in Sproat, in Humana Press 2005 edited by Herdewijn P.; Kap.2: 17-31 and respective downstream processes are, among others,described in Pingoud et al., in IRL Press 1989 edited by Oliver R. W.A.; Kap. 7: 183-208.

Other synthetic procedures are known in the art, e.g. the proceduresdescribed in Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringeet al., 1990, NAR., 18, 5433; Wincott et al., 1995, NAR. 23, 2677-2684;and Wincott et al., 1997, Methods Mol. Bio., 74, 59, may make use ofcommon nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. Themodified (e.g. 2′-O-methylated) nucleotides and unmodified nucleotidesare incorporated as desired.

In some embodiments the oligonucleotides of the present invention aresynthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International Patent Publication No. WO 93/23569; Shabarova et al.,1991, NAR 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16,951; Bellon et al., 1997, Bioconjugate Chem. 8, 204), or byhybridization following synthesis and/or deprotection.

Overlapping pairs of chemically synthesized fragments can be ligatedusing methods well known in the art (e.g., see U.S. Pat. No. 6,121,426).The strands are synthesized separately and then are annealed to eachother in the tube. Then, the double-stranded siRNAs are separated fromthe single-stranded oligonucleotides that were not annealed (e.g.because of the excess of one of them) by HPLC. In relation to themodified siRNA compounds of the present invention, two or more suchsequences can be synthesized and linked together for use in the presentinvention.

The siRNAs compounds of the present invention possess a terminal moietycovalently bound at the 5′-terminus of the antisense strand which ismismatched to a nucleotide in a target mRNA. In various embodiments themoiety at the 5′-terminus of the antisense strand is selected from thegroup consisting of ribouridine, deoxyribouridine, modified ribouridine,modified deoxyribouridine, pseudouracil, deoxypseudouracil,deoxyribothymidine, modified deoxyribothymidine, ribocytosine, modifiedribocytosine, deoxyribocytosine, modified deoxyribocytosine,5-methylribocytosine, modified 5-methylribocytosine,5-methylribouridine, ribo-2-thiouridine, ribo-4-thiouridine, abasicribose moiety and abasic deoxyribose moiety and the moiety at the3′-terminus of the sense strand is selected from a ribonucleotide or amodified ribonucleotide or an unconventional moiety. The siRNAstructures of the present invention are beneficially applied to doublestranded RNA useful in inhibiting or attenuating mammalian andnon-mammalian gene expression.

Pharmaceutical Compositions

While it is possible for the compounds of the present invention to beadministered as the raw chemical, it is preferable to present them as apharmaceutical composition. Accordingly the present invention provides apharmaceutical composition comprising one or more of the modified siRNAcompounds of the invention; and a pharmaceutically acceptable carrier.In some embodiments the pharmaceutical composition comprises two or moremodified siRNA compounds of the invention.

The invention further provides a pharmaceutical composition comprisingat least one compound of the invention covalently or non-covalentlybound to one or more compounds of the invention in an amount effectiveto inhibit a target gene; and a pharmaceutically acceptable carrier. Insome embodiments the modified siRNA compounds are processedintracellularly by endogenous cellular complexes to produce one or moreoligoribonucleotides of the invention.

The invention further provides a pharmaceutical composition comprising apharmaceutically acceptable carrier and one or more of the modifiedsiRNA compounds of the invention in an amount effective to down-regulateexpression in a cell in a mammal of a target gene, the compoundcomprising a sequence which is substantially complementary to thesequence of a target mRNA.

In some embodiments, the modified siRNA compounds according to thepresent invention are the main active component in a pharmaceuticalcomposition. In other embodiments the modified siRNA compounds accordingto the present invention are one of the active components of apharmaceutical composition containing two or more siRNAs, saidpharmaceutical composition further being comprised of one or more siRNAcompounds which target one or more target genes.

The present invention also provides for a process of preparing apharmaceutical composition, which comprises:

providing one or more double stranded modified siRNA compound of theinvention; and

admixing said compound with a pharmaceutically acceptable carrier.

In a preferred embodiment, the modified siRNA compound used in thepreparation of a pharmaceutical composition is admixed with a carrier ina pharmaceutically effective dose. In some embodiments the modifiedsiRNA compound of the present invention is conjugated to a steroid or toa lipid or to another suitable molecule e.g. to cholesterol.

RNA Interference

A number of PCT applications have recently been published that relate tothe RNAi phenomenon. These include: PCT publication WO 00/44895; PCTpublication WO 00/49035; PCT publication WO 00/63364; PCT publication WO01/36641; PCT publication WO 01/36646; PCT publication WO 99/32619; PCTpublication WO 00/44914; PCT publication WO 01/29058; and PCTpublication WO 01/75164.

RNA interference (RNAi) is based on the ability of dsRNA species toenter a cytoplasmic protein complex, where it is then targeted to thecomplementary cellular RNA and specifically degrade it. The RNAinterference response features an endonuclease complex containing asiRNA, commonly referred to as an RNA-induced silencing complex (RISC),which mediates cleavage of single-stranded RNA having a sequencecomplementary to the antisense strand of the siRNA duplex. Cleavage ofthe target RNA may take place in the middle of the region complementaryto the antisense strand of the siRNA duplex (Elbashir et al., GenesDev., 2001, 15(2):188-200). In more detail, longer dsRNAs are digestedinto short (17-29 bp) dsRNA fragments (also referred to as shortinhibitory RNAs, “siRNAs”) by type III RNAses (DICER, DROSHA, etc.;Bernstein et al., Nature, 2001, 409(6818):363-6; Lee et al., Nature,2003, 425(6956):415-9). The RISC protein complex recognizes thesefragments and complementary mRNA. The whole process is culminated byendonuclease cleavage of target mRNA (McManus & Sharp, Nature Rev Genet,2002, 3(10):737-47; Paddison & Hannon, Curr Opin Mol. Ther. 2003,5(3):217-24). (For additional information on these terms and proposedmechanisms, see for example Bernstein et al., RNA 2001, 7(11):1509-21;Nishikura, Cell 2001, 107(4):415-8 and PCT publication WO 01/36646).

The selection and synthesis of siRNA corresponding to known genes hasbeen widely reported; see for example Ui-Tei et al., J BiomedBiotechnol. 2006; 2006: 65052; Chalk et al., BBRC. 2004, 319(1): 264-74;Sioud & Leirdal, Met. Mol. Biol.; 2004, 252:457-69; Levenkova et al.,Bioinform. 2004, 20(3):430-2; Ui-Tei et al., Nuc. Acid Res. 2004,32(3):936-48. For examples of the use of, and production of, modifiedsiRNA see Braasch et al., Biochem., 2003, 42(26):7967-75; Chiu et al.,RNA, 2003, 9(9):1034-48; PCT publications WO 2004/015107 (Atugen); WO02/44321 (Tuschl et al), and U.S. Pat. Nos. 5,898,031 and 6,107,094.

siRNA and RNA Interference

RNA interference (RNAi) is a phenomenon involving double-stranded (ds)RNA-dependent gene specific posttranscriptional silencing. Initialattempts to study this phenomenon and to manipulate mammalian cellsexperimentally were frustrated by an active, non-specific antiviraldefense mechanism which was activated in response to long dsRNAmolecules (Gil et al. Apoptosis, 2000. 5:107-114). Later, it wasdiscovered that synthetic duplexes of 21 nucleotide RNAs could mediategene specific RNAi in mammalian cells, without stimulating the genericantiviral defense mechanisms (see Elbashir et al. Nature 2001,411:494-498 and Caplen et al. PNAS USA 2001, 98:9742-9747). As a result,small interfering RNAs (siRNAs) have become powerful tools in attemptingto understand gene function.

RNA interference (RNAi) in mammals is mediated by small interfering RNAs(siRNAs) (Fire et al, Nature 1998, 391:806) or microRNAs (miRNAs)(Ambros, Nature 2004, 431(7006):350-355; Bartel, Cell 2004, 116(2):281-97). The corresponding process in plants is commonly referred to asspecific post-transcriptional gene silencing (PTGS) or RNA silencing andis also referred to as quelling in fungi.

A siRNA is a double-stranded RNA or modified RNA molecule whichdown-regulates or silences (prevents) the expression of a gene/mRNA ofits endogenous (cellular) counterpart. The mechanism of RNA interferenceis detailed infra.

Several studies have revealed that siRNA therapeutic agents areeffective in vivo in both mammals and in humans. Bitko et al., haveshown that specific siRNA molecules directed against the respiratorysyncytial virus (RSV) nucleocapsid N gene are effective in treating micewhen administered intranasally (Nat. Med. 2005, 11(1):50-55). Recentreviews discussing siRNA therapeutics are available (Batik, et al., J.Mol. Med. 2005, 83:764-773; Dallas and Vlassov, Med. Sci. Monitor 2006,12(4):RA67-74; Chakraborty, Current Drug Targets 2007, 8(3):469-82;Dykxhoorn et al., Gene Therapy 2006. 13:541-552).

Mucke (IDrugs 2007 10(1):37-41) presents a review of currenttherapeutics, including siRNA to various targets, for the treatment ofocular diseases, for example age related macular degeneration (AMD) andglaucoma.

siRNA Structures

The selection and synthesis of siRNA corresponding to known genes hasbeen widely reported; (see for example Ui-Tei et al., J Biomed Biotech.2006; 2006: 65052; Chalk et al., BBRC. 2004, 319(1): 264-74; Sioud &Leirdal, Met. Mol. Biol.; 2004, 252:457-69; Levenkova et al., Bioinform.2004, 20(3):430-2; Ui-Tei et al., NAR. 2004, 32(3):936-48; De Paula etal., RNA 2007, 13:431-56).

For examples of the use of, and production of, modified siRNA see, forexample, Braasch et al., Biochem. 2003, 42(26):7967-75; Chiu et al.,RNA, 2003, 9(9):1034-48; PCT publications WO 2004/015107 (atugen AG) andWO 02/44321 (Tuschl et al). U.S. Pat. Nos. 5,898,031 and 6,107,094,describe chemically modified oligomers. US Patent Publication Nos.2005/0080246 and 2005/0042647 relate to oligomeric compounds having analternating motif and dsRNA compounds having chemically modifiedinternucleoside linkages, respectively.

Other modifications have been disclosed. The inclusion of a 5′-phosphatemoiety was shown to enhance activity of siRNAs in Drosophila embryos(Boutla, et al., Curr. Biol. 2001, 11:1776-1780) and is required forsiRNA function in human HeLa cells (Schwarz et al., Mol. Cell, 2002,10:537-48). Amarzguioui et al., (NAR, 2003, 31(2):589-95) showed thatsiRNA activity depended on the positioning of the 2′-O-methylmodifications. Holen et al (NAR. 2003, 31(9):2401-07) report that ansiRNA having small numbers of 2′-O-methyl modified nucleosides gave goodactivity compared to wild type but that the activity decreased as thenumbers of 2′-O-methyl modified nucleosides was increased. Chiu and Rana(RNA. 2003, 9:1034-48) describe that incorporation of 2′-O-methylmodified nucleosides in the sense or antisense strand (fully modifiedstrands) severely reduced siRNA activity relative to unmodified siRNA.The placement of a 2′-β-methyl group at the 5′-terminus on the antisensestrand was reported to severely limit activity whereas placement at the3′-terminus of the antisense and at both termini of the sense strand wastolerated (Czauderna et al., NAR. 2003, 31(11):2705-16; WO 2004/015107).The molecules of the present invention offer an advantage in that theyare stable and active and are useful in the preparation ofpharmaceutical compositions for treatment of various diseases.

PCT Patent Publication Nos. WO 2008/104978 and WO 2009/044392 to theassignee of the present invention and hereby incorporated by referencein their entirety, disclose novel siRNA structures.

PCT Publication No. WO 2008/050329 and U.S. Ser. No. 11/978,089 to theassignee of the present invention relate to inhibitors of pro-apoptoticgenes, and are incorporated by reference in their entirety.

PCT Patent Publication Nos. WO 2004/111191 and WO 2005/001043 relate tomethods for enhancing RNAi.

The invention provides a method of down-regulating the expression oftarget gene by at least 20%, 30%, 40% or 50% as compared to a control,comprising contacting an mRNA transcript of the target gene with one ormore of the compounds of the invention.

Additionally the invention provides a method of down-regulating theexpression of target gene in a mammal by at least 20%, 30%, 40% or 50%as compared to a control, comprising administering one or more of thecompounds of the invention to the mammal. In a preferred embodiment ofthe invention the mammal is a human.

In various embodiments a double stranded nucleic acid molecule ofStructure (A) is down-regulating the expression of a target gene,whereby the down-regulation of the expression of a target gene isselected from the group comprising down-regulation of gene function(which is examined, e.g. by an enzymatic assay or a binding assay with aknown interactor of the native gene/polypeptide, inter alia),down-regulation of polypeptide product of the gene (which is examined,e.g. by Western blotting, ELISA or immuno-precipitation, inter alia) anddown-regulation of mRNA expression of the gene (which is examined, e.g.by Northern blotting, quantitative RT-PCR, in-situ hybridisation ormicroarray hybridisation, inter alia).

Delivery

The modified siRNA compound of the invention is administered as thecompound per se (i.e. as naked siRNA) or as pharmaceutically acceptablesalt and is administered alone or as an active ingredient in combinationwith one or more pharmaceutically acceptable carrier, solvent, diluent,excipient, adjuvant and vehicle. In some embodiments, the siRNAmolecules of the present invention are delivered to the target tissue bydirect application of the naked molecules prepared with a carrier or adiluent.

The term “naked siRNA” refers to siRNA molecules that are free from anydelivery vehicle that acts to assist, promote or facilitate entry intothe cell, including viral sequences, viral particles, liposomeformulations, lipofectin or precipitating agents and the like. Forexample, siRNA in PBS is “naked siRNA”.

Pharmaceutically acceptable carriers, solvents, diluents, excipients,adjuvants and vehicles as well as implant carriers generally refer toinert, non-toxic solid or liquid fillers, diluents or encapsulatingmaterial not reacting with the active modified siRNA compounds of theinvention and they include liposomes and microspheres. For example, thesiRNA compounds of the invention may be formulated with polyethylenimine(PEI), with PEI derivatives, e.g. oleic and stearic acid modifiedderivatives of branched PEI, with chitosan or withpoly(lactic-co-glycolic acid) (PLGA). Formulating the compositions ine.g. liposomes, micro- or nano-spheres and nanoparticles, may enhancestability and benefit absorption.

Additionally, the compositions may include an artificial oxygen carrier,such as perfluorocarbons (PFCs) e.g. perfluorooctyl bromide(perflubron).

Examples of delivery systems useful in the present invention includeU.S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678;4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196.Many such implants, delivery systems, and modules are well known tothose skilled in the art. In one specific embodiment of this inventiontopical and transdermal formulations are selected.

Accordingly, in some embodiments the siRNA molecules of the inventionare delivered in liposome formulations and lipofectin formulations andthe like and can be prepared by methods well known to those skilled inthe art. Such methods are described, for example, in U.S. Pat. Nos.5,593,972, 5,589,466, and 5,580,859, which are herein incorporated byreference.

Delivery systems aimed specifically at the enhanced and improveddelivery of siRNA into mammalian cells have been developed (see, forexample, Shen et al FEBS Let. 539: 111-114 (2003), Xia et al., Nat.Biotech. 20: 1006-1010 (2002), Reich et al., Mol. Vision. 9: 210-216(2003), Sorensen et al., J. Mol. Biol. 327: 761-766 (2003), Lewis etal., Nat. Gen. 32: 107-108 (2002) and Simeoni et al., NAR 31, 11:2717-2724 (2003)). siRNA has recently been successfully used forinhibition of gene expression in primates; (for details see for example,Tolentino et al., Retina 2004. 24(1):132-138).

Additional formulations for improved delivery of the compounds of thepresent invention can include non-formulated compounds, compoundscovalently bound to cholesterol, and compounds bound to targetingantibodies (Song et al., Antibody mediated in vivo delivery of smallinterfering RNAs via cell-surface receptors, Nat. Biotechnol. 2005.23(6):709-17). Cholesterol-conjugated siRNAs (and other steroid andlipid conjugated siRNAs) can been used for delivery (see for exampleSoutschek et al Nature. 2004. 432:173-177; and Lorenz et al. Bioorg.Med. Chem. Lett. 2004. 14:4975-4977).

The naked siRNA or the pharmaceutical compositions comprising thechemically modified siRNA of the present invention are administered anddosed in accordance with good medical practice, taking into account theclinical condition of the individual patient, the disease to be treated,the site and method of administration, scheduling of administration,patient age, sex, body weight and other factors known to medicalpractitioners.

A “therapeutically effective dose” for purposes herein is thusdetermined by such considerations as are known in the art. The dose mustbe effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art. The siRNA of theinvention can be administered in a single dose or in multiple doses.

In general, the active dose of compound for humans is in the range offrom 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of asingle dose or a one dose per day or twice or three or more times perday for a period of 1-4 weeks or longer.

The modified siRNA compounds of the present invention can beadministered by any of the conventional routes of administration. Themodified siRNA compounds are administered orally, subcutaneously orparenterally including intravenous, intraarterial, intramuscular,intraperitoneally, intraocular, ocular, otic, transtympanic andintranasal administration, intratracheal instillation and intratrachealinhalation, as well as infusion techniques. Implants of the compoundsare also useful.

Liquid forms are prepared for invasive administration, e.g. injection orfor topical or local or non-invasive administration. The term injectionincludes subcutaneous, transdermal, intravenous, intramuscular,intrathecal, intraocular, transtympanic and other parental routes ofadministration. The liquid compositions include aqueous solutions, withand without organic co-solvents, aqueous or oil suspensions, emulsionswith edible oils, as well as similar pharmaceutical vehicles. In aparticular embodiment, the administration comprises intravenousadministration.

In some embodiments the compounds of the present invention areformulated for non-invasive administration. In some embodiments thecompounds of the present invention are formulated as eardrops fortopical administration to the ear. In some embodiments the compounds ofthe present invention are formulated as eye drops for topicaladministration to the surface of the eye. Further information onadministration of the compounds of the present invention can be found inTolentino et al., Retina 2004. 24:132-138; and Reich et al., MolecularVision, 2003. 9:210-216. In addition, in certain embodiments thecompositions for use in the treatments of the present invention areformed as aerosols, for example for intranasal administration. Incertain embodiments the compositions for use in the treatments of thepresent invention are formed as nasal drops, for example for intranasalinstillation.

The therapeutic compositions of the present invention are preferablyadministered into the lung by inhalation of an aerosol containing thesecompositions/compounds, or by intranasal or intratracheal instillationof said compositions. For further information on pulmonary delivery ofpharmaceutical compositions see Weiss et al., Human Gene Therapy 1999.10:2287-2293; Densmore et al., Molecular therapy 1999. 1:180-188; Gautamet al., Molecular Therapy 2001. 3:551-556; and Shahiwala & Misra, AAPSPharmSciTech 2004. 24; 6(3):E482-6. Additionally, respiratoryformulations for siRNA are described in U.S. Patent ApplicationPublication No. 2004/0063654. Respiratory formulations for siRNA aredescribed in US Patent Application Publication No. 2004/0063654.

In certain embodiments, oral compositions (such as tablets, suspensions,solutions) may be effective for local delivery to the oral cavity suchas oral composition suitable for mouthwash for the treatment of oralmucositis.

In a particular embodiment, the modified siRNA compounds of theinvention are formulated for intravenous administration for delivery tothe kidney for the treatment of kidney disorders, e.g. acute renalfailure (ARF), delayed graft function (DGF) and diabetic retinopathy. Itis noted that the delivery of the modified siRNA compounds according tothe present invention to the target cells in the kidney proximal tubulesis particularly effective in the treatment of ARF and DGF.

Delivery of compounds into the brain is accomplished by several methodssuch as, inter alia, neurosurgical implants, blood-brain barrierdisruption, lipid mediated transport, carrier mediated influx or efflux,plasma protein-mediated transport, receptor-mediated transcytosis,absorptive-mediated transcytosis, neuropeptide transport at theblood-brain barrier, and genetically engineering “Trojan horses” fordrug targeting. The above methods are performed, for example, asdescribed in “Brain Drug Targeting: the future of brain drugdevelopment”, W. M. Pardridge, Cambridge University Press, Cambridge, UK(2001).

In addition, in certain embodiments the compositions for use in thetreatments of the present invention are formed as aerosols, for examplefor intranasal administration.

Intranasal delivery for the treatment of CNS diseases has been attainedwith acetylcholinesterase inhibitors such as galantamine and varioussalts and derivatives of galantamine, for example as described in USPatent Application Publication No. 2006003989 and PCT ApplicationsPublication Nos. WO 2004/002402 and WO 2005/102275. Intranasal deliveryof nucleic acids for the treatment of CNS diseases, for example byintranasal instillation of nasal drops, has been described, for example,in PCT Application Publication No. WO 2007/107789.

Methods of Treatment

In one aspect the present invention relates to a method of treating asubject suffering from a disorder associated with target gene expressioncomprising administering to the subject a therapeutically effectiveamount of a modified siRNA compound of the present invention. Inpreferred embodiments the subject being treated is a warm-blooded animaland, in particular, mammal including human.

“Treating a subject” refers to administering to the subject atherapeutic substance effective to ameliorate symptoms associated with adisease, to lessen the severity or cure the disease, to slow down theprogress of the disease, to prevent the disease from occurring or topostpone the onset of the disease. “Treatment” refers to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent a disorder, to delay the onset of the disorderor reduce the symptoms of a disorder. Those in need of treatment includethose already experiencing the disease or condition, those prone tohaving the disease or condition, and those in which the disease orcondition is to be prevented. The compounds of the invention areadministered before, during or subsequent to the onset of the disease orcondition.

A “therapeutically effective dose” refers to an amount of apharmaceutical compound or composition which is effective to achieve animprovement in a subject or his physiological systems including, but notlimited to, improved survival rate, more rapid recovery, improvement orelimination of symptoms, delayed onset of a disorder, slower progress ofdisease and other indicators as are selected as appropriate determiningmeasures by those skilled in the art.

“Respiratory disorder” refers to conditions, diseases or syndromes ofthe respiratory system including but not limited to pulmonary disordersof all types including chronic obstructive pulmonary disease (COPD),emphysema, chronic bronchitis, asthma and lung cancer, inter alia.Emphysema and chronic bronchitis may occur as part of COPD orindependently. In various embodiments the present invention providesmethods and compositions useful in preventing or treating primary graftfailure, ischemia-reperfusion injury, reperfusion injury, reperfusionedema, allograft dysfunction, pulmonary reimplantation response and/orprimary graft dysfunction (PGD) after organ transplantation, inparticular in lung transplantation, in a subject in need thereof.

“Microvascular disorder” refers to any condition that affectsmicroscopic capillaries and lymphatics, in particular vasospasticdiseases, vasculitic diseases and lymphatic occlusive diseases. Examplesof microvascular disorders include, inter alia: eye disorders such asAmaurosis Fugax (embolic or secondary to SLE), apla syndrome, Prot CSand ATIII deficiency, microvascular pathologies caused by IV drug use,dysproteinemia, temporal arteritis, ischemic optic neuropathy (ION),anterior ischemic optic neuropathy (AION), optic neuritis (primary orsecondary to autoimmune diseases), glaucoma, von Hippel Lindau syndrome,corneal disease, corneal transplant rejection cataracts, Eales' disease,frosted branch angiitis, encircling buckling operation, uveitisincluding pars planitis, choroidal melanoma, choroidal hemangioma, opticnerve aplasia; retinal conditions such as retinal artery occlusion,retinal vein occlusion, retinopathy of prematurity, HIV retinopathy,Purtscher retinopathy, retinopathy of systemic vasculitis and autoimmunediseases, diabetic retinopathy, hypertensive retinopathy, radiationretinopathy, branch retinal artery or vein occlusion, idiopathic retinalvasculitis, aneurysms, neuroretinitis, retinal embolization, acuteretinal necrosis, Birdshot retinochoroidopathy, long-standing retinaldetachment; systemic conditions such as Diabetes mellitus, diabeticretinopathy (DR), diabetes-related microvascular pathologies (asdetailed herein), hyperviscosity syndromes, aortic arch syndromes andocular ischemic syndromes, carotid-cavernous fistula, multiplesclerosis, systemic lupus erythematosus, arteriolitis with SS-Aautoantibody, acute multifocal hemorrhagic vasculitis, vasculitisresulting from infection, vasculitis resulting from Behcet's disease,sarcoidosis, coagulopathies, neuropathies, nephropathies, microvasculardiseases of the kidney, and ischemic microvascular conditions, interalia.

Microvascular disorders may comprise a neovascular element. The term“neovascular disorder” refers to those conditions where the formation ofblood vessels (neovascularization) is harmful to the patient. Examplesof ocular neovascularization include: retinal diseases (diabeticretinopathy, diabetic Macular Edema, chronic glaucoma, retinaldetachment, and sickle cell retinopathy); rubeosis iritis; proliferativevitreo-retinopathy; inflammatory diseases; chronic uveitis; neoplasms(retinoblastoma, pseudoglioma and melanoma); Fuchs' heterochromiciridocyclitis; neovascular glaucoma; corneal neovascularization(inflammatory, transplantation and developmental hypoplasia of theiris); neovascularization following a combined vitrectomy andlensectomy; vascular diseases (retinal ischemia, choroidal vascularinsufficiency, choroidal thrombosis and carotid artery ischemia);neovascularization of the optic nerve; and neovascularization due topenetration of the eye or contusive ocular injury. In variousembodiments all these neovascular conditions are treated using thecompounds and pharmaceutical compositions of the present invention.

“Eye disease” refers to conditions, diseases or syndromes of the eyeincluding but not limited to any conditions involving choroidalneovascularization (CNV), wet and dry AMD, ocular histoplasmosissyndrome, angiod streaks, ruptures in Bruch's membrane, myopicdegeneration, ocular tumors, retinal degenerative diseases and retinalvein occlusion (RVO). In various embodiments, conditions disclosedherein, such as DR, which are regarded as either a microvasculardisorder and an eye disease, or both, under the definitions presentedherein, are treated according to the methods of the present invention.

More specifically, the present invention provides methods andcompositions useful in treating a subject suffering from or susceptibleto adult respiratory distress syndrome (ARDS); Chronic obstructivepulmonary disease (COPD); acute lung injury (ALI); Emphysema; DiabeticNeuropathy, nephropathy and retinopathy; diabetic macular edema (DME)and other diabetic conditions; Glaucoma; age related maculardegeneration (AMD); bone marrow transplantation (BMT) retinopathy;ischemic conditions; ocular ischemic syndrome (OIS); kidney disorders:acute renal failure (ARF), delayed graft function (DGF), transplantrejection; hearing disorders (including hearing loss); spinal cordinjuries; oral mucositis; dry eye syndrome and pressure sores;neurological disorders arising from ischemic or hypoxic conditions, suchas hypertension, hypertensive cerebral vascular disease, a constrictionor obstruction of a blood vessel—as occurs in the case of a thrombus orembolus, angioma, blood dyscrasias, any form of compromised cardiacfunction including cardiac arrest or failure, systemic hypotension;stroke, disease, disorders and injury of the CNS, including, withoutbeing limited to, epilepsy, spinal cord injury, brain injury andneurodegenerative disorders, including, without being limited toParkinson's disease, Amyotrophic Lateral Sclerosis (ALS, Lou Gehrig'sDisease), Alzheimer's disease, Huntington's disease and any otherdisease-induced dementia (such as HIV-associated dementia for example).

The present invention relates to compounds, compositions and methodsuseful in the treatment of cancer. The terms “cancer” and “cancerous”refer to or describe the physiological condition in mammals that istypically characterized by unregulated cell growth. Examples of cancerinclude but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia or lymphoid malignancies. Other examples of such cancersinclude kidney or renal cancer, breast cancer, colon cancer, rectalcancer, colorectal cancer, lung cancer including small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung and squamouscarcinoma of the lung, squamous cell cancer (e.g. epithelial squamouscell cancer), cervical cancer, ovarian cancer, prostate cancer, livercancer, bladder cancer, cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer including gastrointestinal cancer,gastrointestinal stromal tumors (GIST), pancreatic cancer, head and neckcancer, glioblastoma, retinoblastoma, astrocytoma, thecomas,arrhenoblastomas, hepatoma, hematologic malignancies includingnon-Hodgkins lymphoma (NHL), multiple myeloma and acute hematologicmalignancies, endometrial or uterine carcinoma, endometriosis,fibrosarcomas, choriocarcinoma, salivary gland carcinoma, vulval cancer,thyroid cancer, esophageal carcinomas, hepatic carcinoma, analcarcinoma, penile carcinoma, nasopharyngeal carcinoma, laryngealcarcinomas, Kaposi's sarcoma, melanoma, skin carcinomas, Schwannoma,oligodendroglioma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma,leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas, Wilm'stumor, as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. “Tumor”, as usedherein, refers to all neoplastic cell growth and proliferation, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues.

Fibrotic disorders include liver fibrosis, cirrhosis, pulmonary fibrosisincluding lung fibrosis (including ILF), kidney fibrosis resulting fromany condition (e.g., CKD including ESRD), peritoneal fibrosis, chronichepatic damage, fibrillogenesis, fibrotic diseases in other organs,abnormal scarring (keloids) associated with all possible types of skininjury accidental and jatrogenic (operations); scleroderma;cardiofibrosis, failure of glaucoma filtering operation; and intestinaladhesions.

Additionally, the invention provides a method of down-regulating theexpression of a target gene by at least 20%, 30%, 40% or 50% as comparedto a control comprising contacting target mRNA with one or more of themodified siRNA compounds of the present invention. In variousembodiments the modified siRNA compound of the present inventiondown-regulates target gene whereby the down-regulation is selected fromthe group comprising down-regulation of gene function, down-regulationof polypeptide and down-regulation of mRNA expression.

The invention provides a method of inhibiting the expression of thetarget gene by at least 20%, 30%, or 40%, preferably by 50%, 60% or 70%,more preferably by 75%, 80% or 90% as compared to a control comprisingcontacting an mRNA transcript of the target gene with one or more of themodified siRNA compounds of the invention.

In one embodiment the modified siRNA compound of the invention inhibitsthe target gene polypeptide, whereby the inhibition is selected from thegroup comprising inhibition of function (which is examined by, forexample, an enzymatic assay or a binding assay with a known interactorof the native gene/polypeptide, inter alia), inhibition of targetprotein (which is examined by, for example, Western blotting, ELISA orimmuno-precipitation, inter alia) and inhibition of target mRNAexpression (which is examined by, for example, Northern blotting,quantitative RT-PCR, in-situ hybridization or microarray hybridization,inter alia).

In additional embodiments the invention provides a method of treating asubject suffering from or susceptible to any disease or disorderaccompanied by an elevated level of a mammalian or non-mammalian targetgene, the method comprising administering to the subject a modifiedsiRNA compound or composition of the invention in a therapeuticallyeffective dose thereby treating the subject.

The present invention relates to the use of compounds whichdown-regulate the expression of a mammalian target gene particularly tomodified RNA compounds according to structure (A) in the treatment ofthe following diseases or conditions in which inhibition of theexpression of the mammalian target gene is beneficial: ARDS; COPD; ALI;Emphysema; Diabetic Neuropathy, nephropathy and retinopathy; DME andother diabetic conditions; Glaucoma; AMD; BMT retinopathy; ischemicconditions including stroke; OIS; Neurodegenerative disorders such asParkinson's disease, Alzheimer's disease, ALS; kidney disorders: ARF,DGF, transplant rejection; hearing disorders; spinal cord injuries; oralmucositis; cancer including hematopoietic and solid tumor cancer, dryeye syndrome and pressure sores. In another embodiment the compounds ofthe present invention are useful in organ storage and/or preservationbefore transplant.

By “exposure to a toxic agent” is meant that the toxic agent is madeavailable to, or comes into contact with, a mammal. A toxic agent can betoxic to the nervous system. Exposure to a toxic agent can occur bydirect administration, e.g., by ingestion or administration of a food,medicinal, or therapeutic agent, e.g., a chemotherapeutic agent, byaccidental contamination, or by environmental exposure, e.g., aerial oraqueous exposure.

In other embodiments the chemically modified siRNA compounds and methodsof the invention are useful for treating or preventing the incidence orseverity of other diseases and conditions in a subject. These diseasesand conditions include, but are not limited to stroke and stroke-likesituations (e.g. cerebral, renal, cardiac failure), neuronal cell death,brain injuries with or without reperfusion, spinal cord injury, chronicdegenerative diseases e.g. neurodegenerative disease including,Alzheimer's disease, Parkinson's disease, Huntington's disease, multiplesclerosis, spinobulbar atrophy, prion disease and apoptosis resultingfrom traumatic brain injury (TBI). In an additional embodiment, thecompounds and methods of the invention are directed to providingneuroprotection, and or cerebroprotection.

Without limitation a target gene is selected from the group consistingof p53 (TP53), TP53BP2, LRDD, CYBA, ATF3, CASP2 (Caspase 2), NOX3, HRK;C1QBP, BNIP3, MAPK8; Rac1, GSK3B, CD38, STEAP4, BMP2a; GJA1, TYROBP,CTGF, SPP1, RTN4R, ANXA2, RHOA, DUOX1, SLC5A1, SLC2A2, AKR1B1, SORD,SLC2A1, MME, NRF2, SRM, REDD2 (RTP801L), REDD1 (RTP801), NOX4, MYC,PLK1, ESPL1, HTRA2, KEAP1, p66, ZNHIT1, LGALS3, CYBB (NOX2), NOX1,NOXO1, ADRB1, HI 95, ARF1, ASPP1, SOX9, FAS, FASLG, Human MLL, AF9,CTSD, CAPNS1, CD80, CD86, HEST, HESS, CDKN1B, ID1, ID2, ID3, CDKN2A,Caspase 1, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7,Caspase 8, Caspase 9, Caspase 10, Caspase 12, Caspase 14, Apaf-1, Nod1,Nod2, Ipaf, DEFCAP, RAIDD, RICK, Bcl10, ASC, TUCAN, ARC, CLARP, FADD,DEDD, DEDD2, Cryopirin, PYC1, Pyrin, TRADD, UNC5a, UNC5b, UNC5c, ZUD,p84N5, LRDD, CDK1, CDK2, CDK4, CDK5, CDK9, PITSLRE A, CHK2, LATS1, Prk,MAP4K1, MAP4K2, STK4, SLK, GSK3alpha, GSK3beta, MEKK1, MAP3K5 (Ask1),MAP3K7, MAP3K8, MAP3K9, MAP3K10, MAP3K11, MAP3K12, DRP-1, MKK6, p38,JNK3, DAPK1, DRAK1, DRAK2, IRAK, RIP, RIP3, RIPS, PKR, IRE1, MSK1,PKCalpha, PKCbeta, PKCdelta, PKCepsilon, PKCeta, PKCmu, PKCtheta,PKCzeta, CAMK2A, HIPK2, LKB1, BTK, c-Src, FYN, Lck, ABL2, ZAP70, TrkA,TrkC, MYLK, FGFR2, EphA2, AATYK, c-Met, RET, PRKAA2, PLA2G2A, SMPD1,SMPD2, SPP1, FAN, PLCG2, IP6K2, PTEN, SHIP, AIF, AMID, Cytochrome c,Smac, HtrA2, TSAP6, DAP-1, FEM-, DAP-3, Granzyme B, DIO-1, DAXX, CAD,CIDE-A, CIDE-B, Fsp27, Apel, ERCC2, ERCC3, BAP31, Bit1, AES, Huntingtin,HIP1, hSir2, PHAP1, GADD45b, GADD34, RAD21, MSH6, ADAR, MBD4, WW45, ATM,mTOR, TIP49, diubiquitin/FAT10, FAF1, p193, Scythe/BAT3, Amida, IGFBP-3,TDAG51, MCG10, PACT, p52/RAP, ALG2, ALG3, presenelin-1, PSAP, AIP1/Alix,ES18, mda-7, p14ARF, ANT1, p33ING1, p33ING2, p53AIP1, p53DINP1,MGC35083, NRAGE, GRIM19, lipocalin 2, glycodelin A, NADE, Porimin,STAG1, DAB2, Galectin-7, Galectin-9, SPRC, FLJ21908, WWOX, XK, DKK-1,Fzd1, Fzd2, SARP2, axin 1, RGS3, DVL1, NFkB2, IkBalpha, NF-ATC1,NF-ATC2, NF-ATC4, zf3/ZNF319, Egr1, Egr2, Egr3, Sp1, TIEG, WT1, Zac1,Icaros, ZNF148, ZK1/ZNF443, ZNF274, WIG1, HIVEP1, HIVEP3, Fliz1, ZPR9,GATA3, TR3, PPARG, CSMF, RXRa, RARa, RARb, RAR9, T3Ra, Erbeta, VDR,GR/GCCR, p53, p73alpha, p63(human [ta alpha, ta beta, ta gamma, daalpha, a beta, da gamma], 53BP2, ASPP1, E2F1, E2F2, E2F3, HIF1 alpha,TCF4, c-Myc, Max, Mad, MITF, Id2, Id3, Id4, c-Jun, c-Fos, ATF3, NF-IL6,CHOP, NRF1, c-Maf, Bach2, Msx2, Csx, Hoxa5, Ets-1, PU1/Spi1, Ets-2,ELK1, TEL1, c-Myb, TBX5, IRF1, IRF3, IRF4, IRF9, AP-2 1pha, FKHR,FOXO1A, FKHRL1, FOXO3a, AFX1, MLLT7, Tip60, BTG1, AUF1, HNRPD, TIAL,NDG1, PCBP4, MCG10, FXR2, TNFR2, LTbR, CD40, CD27, CD30, 4-1BB,TNFRSF19, XEDAR, Fn14, OPG, DcR3, FAS, TNFR1, WSL-1, p75NTR, DR4, DR5,DR6, EDAR, TNF 1pha, FAS ligand, TRAIL, Lymphotoxin alpha, Lymphotoxinbeta, 4-1BBL, RANKL, TL1, TWEAK, LIGHT, APRIL, IL-1-alpha, IL-1-beta,IL-18, FGF8, IL-2, IL-21, IL-5, IL-4, IL-6, LIF, IL-12, IL-7, IL-10,IL-19, IL-24, IFN alpha, IFN beta, IFN gamma, M-CSF, Prolactinm, TLR2,TLR3, TLR4, MyD88, TRIF, RIG-1, CD14, TCR alpha, CD3 gamma, CD8, CD4,CD7, CD19, CD28, CTLA4, SEMA3A, SEMA3B, HLA-A, HLA-B, HLA-L,HLA-Dmalpha, CD22, CD33, CALL, DCC, ICAM1, ICAM3, CD66a, PVR, CD47, CD2,Thy-1, SIRPal, CD5, E-cadherin, ITGAM, ITGAV, CD18, ITGB3, CD9, IgE Fc Rbeta, CD82, CD81, PERP, CD24, CD69, KLRD1, galectin 1, B4GALT1, Clqalpha, C5R1, MIPlalpha, MIPlbeta, RANTES, SDF1, XCL1, CCCKR5, OIAS/OAS1,INDO, MxA, IFI16, AIM2, iNOS, HB-EGF, HGF, MIF, TRAF3, TRAF4, TRAF6,PAR-4, IKKGamma, FIP2, TXBP151, FLASH, TRF1, IEX-1S, Dok1, BLNK, CIN85,Bif-1, HEFT, Vav1, RasGRP1, POSH, Rac1, RhoA, RhoB, RhoC, ALG4, SPP1,TRIP, SIVA, TRABID, TSC-22, BRCA1, BARD1, 53BP1, MDC1, Mdm4, Siah-1,Siah-2, RoRet, TRIM35, PML, RFWD1, DIPJ, Socsl, PARC, USP7, CYLD,SERPINH1 (HSP47). Other useful target genes are genes of microbialorigin.

Combination Therapy

The methods of treating the diseases disclosed herein includeadministering a modified double stranded nucleic acid molecule disclosedherein in conjunction or in combination with an additional inhibitor, asubstance which improves the pharmacological properties of the modifiedsiRNA compound, or an additional compound known to be effective in thetreatment of a subject suffering from or susceptible to any of thehereinabove mentioned diseases and disorders, including microvasculardisorder, eye disease and condition (e.g. macular degeneration), hearingimpairment (including hearing loss), respiratory disorder, kidneydisorder, organ transplantation, neurodegenerative disorder, spinal cordinjury, brain injury, angiogenesis- and apoptosis-related condition.

The present invention thus provides in another aspect, a pharmaceuticalcomposition comprising a combination of a therapeutic modified siRNAcompound of the invention together with at least one additionaltherapeutically active agent. By “in conjunction with” or “incombination with” is meant prior to, simultaneously or subsequent to.Accordingly, the individual components of such a combination areadministered either sequentially or simultaneously from the same orseparate pharmaceutical formulations.

Combination therapies comprising known treatments for treatingmicrovascular disorders, eye disease and conditions (e.g. maculardegeneration), hearing impairments (including hearing loss), respiratorydisorders, kidney disorders, organ transplantation, neurodegenerativedisorders (e.g. spinal cord injury), angiogenesis- and apoptosis-relatedconditions, in conjunction with the modified siRNA compounds andtherapies described herein are considered part of the current invention.

Accordingly, in another aspect of present invention, an additionalpharmaceutically effective compound is administered in conjunction withthe pharmaceutical composition of the invention. In addition, themodified siRNA compounds of the invention are used in the preparation ofa medicament for use as adjunctive therapy with a second therapeuticallyactive compound to treat such conditions. Appropriate doses of knownsecond therapeutic agents for use in combination with a chemicallymodified siRNA compound of the invention are readily appreciated bythose skilled in the art.

In some embodiments the combinations referred to above are presented foruse in the form of a single pharmaceutical formulation.

The administration of a pharmaceutical composition comprising any one ofthe pharmaceutically active siRNA conjugates according to the inventionis carried out by any of the many known routes of administration,including intravenously, intra-arterially, subcutaneously,intra-peritoneally or intra-cerebrally, as determined by a skilledpractitioner. Using specialized formulations, it is possible toadminister the compositions orally or via inhalation or via intranasalinstillation. In some embodiments a compound of the present invention isformulated for topical administration, including as eardrops, eye drops,dermal formulation, transdermal formulation and the like.

By “in conjunction with” is meant that the additional pharmaceuticallyeffective compound is administered prior to, at the same time as, orsubsequent to administration of the compounds or the pharmaceuticalcompositions of the present invention. The individual components of sucha combination referred to above, therefore, can be administered eithersequentially or simultaneously from the same or separate pharmaceuticalformulations. As is the case for the present modified siRNA compounds, asecond therapeutic agent can be administered by any suitable route, forexample, by oral, buccal, inhalation, sublingual, rectal, vaginal,transurethral, nasal, otic, ocular, topical, percutaneous (i.e.,transdermal), or parenteral (including intravenous, intramuscular,subcutaneous, and intracoronary) administration.

In some embodiments, a modified siRNA compound of the invention and thesecond therapeutic agent are administered by the same route, eitherprovided in a single composition as two or more different pharmaceuticalcompositions. However, in other embodiments, a different route ofadministration for the modified siRNA compound of the invention and thesecond therapeutic agent is either possible or preferred. Personsskilled in the art are aware of the best modes of administration foreach therapeutic agent, either alone or in combination.

In various embodiments, the modified siRNA compounds of the presentinvention are the main active component in a pharmaceutical composition.

In another aspects, the present invention provides a pharmaceuticalcomposition comprising two or more siRNA molecules for the treatment ofa disease and for any of the diseases and conditions mentioned herein.In some embodiments the two or more siRNA molecules or formulationscomprising said molecules are admixed in the pharmaceutical compositionin amounts that generate equal or otherwise beneficial activity. Incertain embodiments the two or more siRNA molecules are covalently ornon-covalently bound, or joined together by a nucleic acid linker of alength ranging from 2-100, preferably 2-50 or 2-30 nucleotides.

In some embodiments the pharmaceutical compositions of the inventionfurther comprise one or more additional siRNA molecule, which targetsone or more additional gene. In some embodiments, simultaneousinhibition of said additional gene(s) provides an additive orsynergistic effect for treatment of the diseases disclosed herein.

The treatment regimen according to the invention is carried out, interms of administration mode, timing of the administration, and dosage,so that the functional recovery of the patient from the adverseconsequences of the conditions disclosed herein is improved or so as topostpone the onset of a disorder.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology used is intended to be in the natureof words of description rather than of limitation.

Modifications and variations of the present invention are possible inlight of the above teachings. It is, therefore, to be understood thatwithin the scope of the appended claims, the invention can be practicedotherwise than as specifically described.

The present invention is illustrated in detail below with reference toexamples, but is not to be construed as being limited thereto.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

EXAMPLES

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe claimed invention in any way.

Standard molecular biology protocols known in the art not specificallydescribed herein are generally followed essentially as in Sambrook etal., Molecular cloning: A laboratory manual, Cold Springs HarborLaboratory, New-York (1989, 1992), and in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1988), and as in Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1989) and as in Perbal, APractical Guide to Molecular Cloning, John Wiley & Sons, New York(1988), and as in Watson et al., Recombinant DNA, Scientific AmericanBooks, New York and in Birren et al (eds) Genome Analysis: A LaboratoryManual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York(1998) and methodology as set forth in U.S. Pat. Nos. 4,666,828;4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein byreference. Polymerase chain reaction (PCR) was carried out generally asin PCR Protocols: A Guide To Methods And Applications, Academic Press,San Diego, Calif. (1990). In situ (In cell) PCR in combination with FlowCytometry is useful for detection of cells containing specific DNA andmRNA sequences (Testoni et al., Blood 1996, 87:3822.) Methods ofperforming RT-PCR are also well known in the art.

Example 1 Generation of Sequences for siRNAs to Target Genes andProduction of the Modified siRNA Compounds

Using proprietary algorithms and the known sequence of any gene,optionally the pro-apoptotic genes disclosed herein, t19-mer sequencesof many potential siRNAs were generated. In addition to the algorithm,some 23-mer oligomer sequences are generated by 5′ and/or 3′ extensionof the 19-mer sequences. The antisense strand sequences that weregenerated using this method are fully or substantially complementary toa section of target mRNA sequence. In some embodiments the antisensesequence is fully complementary to a section of the corresponding mRNAsequence. For generating the modified siRNA compounds of the invention,the nucleotide at the 5′ terminal position of the antisense strand (N)x(position 1; N¹) was substituted to generate a double stranded nucleicacid molecule of embodiments of structure (A). In other examples, thenucleotide at the 5′ terminal position of the antisense strand (N)x andthe nucleotide at the 3′ terminal position of the sense strand (N′)ywere substituted to generate the double stranded nucleic acid moleculeof embodiments of structure (A).

Table A provides nucleotide sequences of sense and antisense strands ofcompounds used in some of examples. Table A1 provides exemplary doublestranded nucleic acid molecules that were generated as described toprovide modified siRNA compounds according to embodiments of structure(A). The compounds shown herein are not meant to be limiting and anytarget RNA my be used to identify double stranded nucleic acid moleculesas described herein. Table A2 provides modified siRNA compounds thatwere generated for comparative testing. The modified siRNA compounds inTables A1 and A2 target TLR2 (Homo sapiens toll-like receptor 2 (TLR2),mRNA, gi|68160956|ref|NM_(—)003264.3|, SEQ ID NO:4) gene and CAPNS1(calpain, small subunit 1 variant gi|51599152|ref|NM_(—)001749.2|; SEQID NO:1 or calpain, small subunit 1 variant 2gi|51599150|ref|NM_(—)001003962.1|, SEQ ID NO:2) gene. Additionalcompounds target human RhoA mRNA (ras homolog gene family, member A(RhoA), mRNA gi|50593005 |ref|NM_(—)001664.2|, SEQ ID NO:3). mRNAsequences of mammalian target genes are available, for example, on theNCBI web site [http://www.ncbi.nlm.nih.gov/].

TABLE A  dsRNA SEQ SEQ molecule ID SENSE ID ANTISENSE CAPNS1_23 5GAGAUGACAUGGAGG 10 CUGACCUCCAUGUC UCAG AUCUC TLR2_16 6 GAGUGGUGCAAGUAU11 GUUCAUACUUGCAC GAAC CACUC TLR2_37 7 GGUGGAGAACCUUAU 12 GACCAUAAGGUUCUGGUC CCACC TLR2_46 8 AGAUAAUGAACACCA 13 GUCUUGGUGUUCAU AGAC UAUCURHOA_61 9 GAUCUUCGGAAUGAU 14 UCUCAUCAUUCCGA GAGA AGAUC

TABLE A1  Exemplary TLR2 double stranded molecules of the invention(N′)y siRNA Compound (N)x TLR2_16_S1016 SEN 5′ G A G U G G U G C A A G UA U G A A G 3′ AS 5′ UUUCAUACUUGCACCACUC 3′ TLR2_16_S1018 SEN 5′ G A G UG G U G C A A G U A U G A A G 3′ AS 5′ CUUCAUACUUGCACCACUC 3′TLR2_16_S1019 SEN 5′ G A G U G G U G C A A G U A U G A A G 3′ AS 5′dUUUCAUACUUGCACCACUC 3′ TLR2_16_S1020 SEN 5′ G A G U G G U G C A A G U AU G A A G 3′ AS 5′ dBUUCAUACUUGCACCACUC 3′ TLR2_16_S1022 SEN 5′ G A G UG G U G C A A G U A U G A A U 3′ AS 5′ UUUCAUACUUGCACCACUC 3′TLR2_16_S1024 SEN 5′ G A G U G G U G C A A G U A U G A A U 3′ AS 5′CUUCAUACUUGCACCACUC 3′ TLR2_16_S1025 SEN 5′ G A G U G G U G C A A G U AU G A A U 3′ AS 5′ dUUUCAUACUUGCACCACUC 3′ TLR2_16_S1026 SEN 5′ G A G UG G U G C A A G U A U G A A U 3′ AS 5′ dBUUCAUACUUGCACCACUC 3′TLR2_16_S1028 SEN 5′ G A G U G G U G C A A G U A U G A A C 3′ AS 5′CUUCAUACUUGCACCACUC 3′ TLR2_16_S1029 SEN 5′ G A G U G G U G C A A G U AU G A A C 3′ AS 5′ dUUUCAUACUUGCACCACUC 3′ TLR2_16_S1030 SEN 5′ G A G UG G U G C A A G U A U G A A C 3′ AS 5′ dBUUCAUACUUGCACCACUC 3′TLR2_16_S1032 SEN 5′ G A G U G G U G C A A G U A U G A A A 3′ AS 5′CUUCAUACUUGCACCACUC 3′ TLR2_16_S1033 SEN 5′ G A G U G G U G C A A G U AU G A A A 3′ AS 5′ dUUUCAUACUUGCACCACUC 3′ TLR2_16_S1034 SEN 5′ G A G UG G U G C A A G U A U G A A A 3′ AS 5′ dBUUCAUACUUGCACCACUC 3′TLR2_16_S941 SEN 5′ G A G U G G U G C A A G U A U G A A C 3′ AS 5′UUUCAUACUUGCACCACUC 3′ TLR2_16_S938 SEN 5′ G A G U G G U G C A A G U A UG A A A 3′ AS 5′ UUUCAUACUUGCACCACUC 3′ CAPNS1_23_S938 SEN 5′ G A G A UG A C A U G G A G G U C A A 3′ AS 5′ UUGACCUCCAUGUCAUCUC 3′CAPNS1_23_S898 SEN 5′ G A G A U G A C A U G G A G G U C A A 3′ AS 5′ U UG A C C U C C A U G U C A U C U C -dTdT 3′ CAPNS1_23_S939 SEN 5′ G A G AU G A C A U G G A G G U CA A  3′ AS 5′ CUGACCUCCAUGUCAUCUC 3′

TABLE A2  Compounds that were synthesized for comparative testing (N′)ysiRNA Compound (N)x TLR2_16_S1015 SEN 5′ G A G U G G U G C A A G U A U GA A G 3′ AS 5′ GUUCAUACUUGCACCACUC 3′ TLR2_16_S1017 SEN 5′ G A G U G G UG C A A G U A U G A A G 3′ AS 5′ AUUCAUACUUGCACCACUC 3′ TLR2_16_S1021SEN 5′ G A G U G G U G C A A G U A U G A A U 3′ AS 5′GUUCAUACUUGCACCACUC 3′ TLR2_16_S1023 SEN 5′ G A G U G G U G C A A G U AU G A A U 3′ AS 5′ AUUCAUACUUGCACCACUC 3′ TLR2_16_S1027 SEN 5′ G A G U GG U G C A A G U A U G A A C 3′ AS 5′ AUUCAUACUUGCACCACUC 3′TLR2_16_S1031 SEN 5′ G A G U G G U G C A A G U A U G A A A 3′ AS 5′AUUCAUACUUGCACCACUC 3′ TLR2_16_S73 SEN 5′ G A G U G G U G C A A G U A UG A A C 3′ AS 5′ GUUCAUACUUGCACCACUC 3′ TLR2_16_S939 SEN 5′ G A G U G GU G C A A G U A U G A A A 3′ AS 5′ GUUCAUACUUGCACCACUC 3′ CAPNS1_23_S73SEN 5′ G A G A U G A C A U G G A G G U C A G 3′ AS 5′CUGACCUCCAUGUCAUCUC 3′ CAPNS1_23_S867 SEN 5′ G A G A U G A C A U G G A GG U C A G 3′ AS 5′ C U G A C C U C C A U G U C A U C U C -dTdT 3′

Table A3 hereinbelow provides a code of the modifiednucleotides/unconventional moieties utilized in preparing the siRNAoligonucleotides of Tables A1 and A2.

TABLE A3 Code of the modified nucleotides/unconventional moieties asused in the Tables herein. Code Modification dT thymidine-3′-phosphate A2′-O-methyladenosine-3′-phosphate C 2′-O-methylcytidine-3′-phosphate G2′-O-methylguanosine-3′-phosphate U 2′-O-methyluridine-3′-phosphate dBInverted abasic deoxyribose-3′-phosphate dUdeoxyribouridine-3′-phosphate LdC L-deoxyribocytidine-3′-phosphate(enantiomeric dC) C3 C3—OH C3C3 C3Pi-C3OH

Example 2 In Vitro Testing of Modified siRNA Compounds

Protocol for Testing siRNA Activity

The modified siRNA compounds according to the present invention aretested for activity as follows: About 1×10⁵ rat REF52 (for TLR2 siRNAactivity) or 0.6×10⁵ human PC3 cells (for CAPNS1 siRNA activity) wereseeded per well in 6 wells plate (30-70% confluent). After about 24 hincubation, cells were transfected with dsRNA oligos (see Tables A4 andA5 below), using Lipofectamine™ 2000 (Invitrogene) at finalconcentrations of 1-40 nM for TLR2 dsRNA and at final concentrations of5-40 nM for CAPNS1 dsRNA

TABLE A4 TLR2_16 siRNA compounds (SEQ ID NOS: 6 and 11)

TABLE A5 CAPNS1_23 siRNA compounds (SEQ ID NOS: 5 and 10)

As positive control for cell transfection efficiency, REDD14-Cy3.5labeled oligos were used. As negative control for siRNA activityscrambled (CNL) oligos were used (same concentration as mentioned insection 1.2). Transfection efficiency was tested by fluorescentmicroscopy, 24 hrs following cell transfection.

siRNA Sample preparation: For each transfected well 3 μlLipofectamine2000 reagent was diluted in 250 μl serum free medium, andincubate for 5 min at RT.

siRNA molecules were prepared as follows:

siRNA working solution:

REDD14-Cy3.5 stock 1.5×10⁶ nM (dilute 1:150 to have final concentrationof 10 μM with PBS)

CNL stock 1.6×10⁶ nM (dilute 1:160 to have final concentration of 10 μMwith PBS)

Target genes stock 100 μM (dilute 1:10 to have final concentration of 10μM with PBS)

Samples were diluted to final concentration for TLR2, CAPNS1 and CNLsiRNA oligos as mentioned above (section 1.2 and 1.4) and 20 nM ofREDD14-Cy3.5 siRNA in 250 DMEM medium, calculated to final volume of 2ml per well. Lipofectamine™ 2000 was combined with siRNA (1:1 volume),sample was mixed gently and incubated at room temperature for 20minutes.

Transfection: The Lipofectamine/siRNA complex was added onto the cells(500 μl per well), the plate was rocked back and forth (2 ml finalvolume in each well). Cells were incubated at 37° C. in a CO₂ incubator.

Quantification of mRNA levels by qPCR: 48 hrs following cellstransfection, cells were harvested and RNA was isolated using EZ-RNA™kit [Biological Industries (#20-410-100). cDNA Synthesis was performedand mRNA level of was measured by qPCR. Measured mRNA quantities werenormalized to the mRNA quantity of the reference gene peptidylprolylisomerase A (cyclophilin A; CycloA)

siRNA Activity Results:

TLR2_(—)16 siRNA activity: qPCR analysis of rat TLR2 expression in REF52cells expressing endogenous gene, following TLR2_(—)16 siRNAtransfection (data in Table A6 demonstrate residual (% ofCtrl-un-transfected cells) rat TLR2 expression in REF52 cells.

TABLE A6

The most active compound was TLR2_(—)16_S938 which has AS mismatch totarget mRNA and funny complementary duplex. In TLR2_(—)16_S938 N¹=U andN²=A and N¹ is mismatched to C in target mRNA.

CAPNS1_(—)23 siRNA Activity

qPCR analysis of CAPNS1 expression in human PC-3 cells expressingendogenous gene, following CAPNS1_(—)23 siRNA transfection (data inTable A7 demonstrate residual (% of Ctrl-un-transfected cells) humanCAPNS1 expression in PC-3 cells.

TABLE A7

The most active compound was CAPNS1_(—)23 S938 which has AS mismatch totarget mRNA and fully complementary duplex. In CAPNS1_(—)23 S938 N¹=Uand N²=A and N¹ is mismatched to C in target mRNA.

The test compounds according to Structure (A) structures, are at least10% more active than the control compounds, wherein the antisense strandis fully complementary to the target mRNA.

On-Target and Off-Target Testing of Double Stranded RNA Molecules

The purpose of this study was to assess the on-target activity andpotential off-target activity of control (unmodified) and test(chemically modified) double stranded nucleic acid molecules.

The two strands of a siRNA molecule have sequences with configurationsthat are sense and antisense with respect to the target gene mRNA.Within a cell, the antisense strand of siRNA, known as the guide strand(GS) is loaded into the RNA-induced silencing complex (RISC) and servesto guide the RNAi machinery to complementary sequences in target mRNA.The sense strand, known as the passenger strand (PS), is destroyed. Whenexact complementarity exists between the GS and the target mRNA thelatter is cleaved by the RNaseH-like slicer activity of RISC.

In some cases a siRNA molecule may down-regulate unintended genes whosetranscripts possess complementarity to the GS seed region (nucleotidesat positions 2-8 [5′>3′]) in the 3′-UTR. Without wishing to be bound totheory, this off-target effect may be mediated by a mechanism similar tothat of target silencing by microRNAs (miRNAs). Another type ofoff-target activity of siRNA may occur due to loading of the sensestrand (PS) into RISC. The unintended off-target effects of syntheticsiRNAs can be reduced or abrogated by chemical modification of theinitial siRNA sequence in the siRNA duplex. The test molecules weredesigned accordingly.

Test molecules were assessed for both on-target activity (activity totarget mRNA) and off-target activity (activity to mRNA other than targetmRNA) in the “guide-seed-sequence-and-passenger-strand-based activityassay” using the psiCHECK™ (Promega) plasmid constructs. The activity oftest and control molecules was tested against either a full targetsequence (nucleotide sequence fully complementary to the whole 19-basesequence of either the GS or PS of test molecule) or the seed-targetsequence (sequence complementary to nucleotides 1-8 [5′>3′] of eitherthe GS or PS of test molecule).

The test molecules were at least as active against the GS full targetsite than was the non-modified control siRNA. Test molecules wereinactive towards the PS full target site, whereas the controlnon-modified siRNA demonstrated activity towards the same site. BothsiRNAs were inactive against the GS seed-target sequence and the PSseed-target sequence sites.

Guide strand (GS) refers to the antisense strand of a double strandedRNA that is able to enter the RISC complex and guide silencing of thetarget RNA.

Passenger strand (PS) refers to the sense strand of a double strandedRNA.

Seed sequence refers to nucleotides 2-8 (5′>3′) of the GS and relevantfor the off-target recognition.

CM (complete match) refers to a synthetic DNA fragment with nucleotidesequence fully complementary to the guide strand of the double strandedRNA molecule. This DNA fragment is cloned in 3′UTR of a reporter geneand serves as a target for RNA silencing. (Castanotto & Rossi (2009).Nature, 22:426-33)

SM (seed match) refers to a synthetic DNA fragment with nucleotidesequence with full complementarity to the nucleotides 1-8 (5′>3′) of theguide strand of the test molecule siRNA (1st nucleotide+seed). This DNAfragment is cloned in 3′UTR of a reporter gene and serves as a targetfor the seed-based “off-target” silencing.

The psiCHECK™ system enables evaluation of the GS (antisense) and the PS(sense strand) to elicit targeted and off-targeted effects, bymonitoring the changes in expression levels of their target sequences.Four psiCHECK™-2-based (Promega) constructs were prepared for theevaluation of target activity and potential off-target activity of eachtest molecule GS and PS strands. In each of the constructs one copy orthree copies of either the full target or the seed-target sequence, oftest molecule PS or GS, was cloned into the multiple cloning sitelocated downstream of the Renilla luciferase translational stop codon inthe 3′-UTR region. The resulting vectors were termed:

1-GS-CM (guide strand, complete-match) vector containing one copy orthree copies of the full target sequence (nucleotide sequence fullycomplementary to the whole 19-base sequence of the GS of the testmolecule);

2-PS-CM (passenger strand, complete-match) vector containing one copy orthree copies of the full target sequence (nucleotide sequence fullycomplementary to the whole 19-base sequence of the PS of the testmolecule);

3-GS-SM (guide strand, seed-match) vector containing one copy or threecopies of the seed region target sequence (sequence complementary tonucleotides 1-8 of the GS of the test molecule);

4-PS-SM (passenger strand, seed-match) vector containing one copy orthree copies of the seed region target sequence (sequence complementaryto nucleotides 1-8 of the PS of the test molecule).

The target sequences, with or without nucleotide substitutions atposition 19 (position 1 of AS) were cloned downstream to the codingregion of the Renilla luciferase reporter gene.

The RNAi or seed-mediated activity of a test molecule toward any ofthese sequences results either in cleavage and subsequent degradation ofthe fusion mRNA (GS) or in translational inhibition (PS). In both casesprotein expression is attenuated.

Cloning of Test Molecule Gs and Ps Seed and Full Target Sites

A single copy of the relevant target cloned in the 3'UTR of the reportermRNA, Renilla Luciferase in the psiCHECK™-2 (Promega) vector. There aremultiple cloning sites in the vector. Typical cloning sites that wereused are XhoI and NotI. Vector was prepared for cloning using standardmolecular biology techniques. Each strand of CM and SM was chemicallysynthesized and annealed by heating to 100° C. and cooled to roomtemperature. Ligation was carried out for 3 hours using standardmolecular biology techniques. Ligated plasmids were transformed into E.coli DH5a cells

Resulting colonies were screened for presence of plasmid constructs bycolony-PCR using relevant primers. Each of the plasmids (vectors) waspurified from one positive colony and its sequence was verified.

Transfection of Vectors into Human HeLa Cells.

About 1.3−2×10⁶ human HeLa cells (ATCC, Cat#CCL-2) were inoculated per10 cm dish. Cells were then incubated in 37±1° C., 5% CO₂ for 24 hours.Growth medium was replaced one day post inoculation by 8 mL fresh growthmedium prepared. Each cell-containing plate was transfected with one ofthe vectors, using Lipofectamine™ 2000 reagent (Invitrogen) as follows:

In an Eppendorf tube, 15 μL Lipofectamine™ 2000 reagent was diluted in1000 μL DMEM medium and incubated for 5 minutes at room temperature(RT). In a second Eppendorf tube, a vector was diluted to reach a finalconcentration of 15 μg in 1000 μL DMEM medium. The dilutedLipofectamine™ 2000 reagent was mixed gently with the diluted DNA vectorsample and incubated for 20-40 minutes at RT. Following incubation,DNA/Lipofectamine™ 2000 was added (to reach a 2000 μL final volume) tothe cells. The plates were gently rocked. Plates were incubated for 5hours at 37±1° C. and 5% CO₂. Following a 5-hour incubation, cells werere-plated in a 96-well plate at final concentration of 5×10³ cells perwell in 80 μL growth medium. 16 hours later, cells were transfected withtest or control molecules using Lipofectamine™ 2000. Duplicatetransfections of each siRNA concentration were performed, as describedbelow:

Lipofectamine™ 2000 was prepared in excess to suffice for 170 wells: 85μL of Lipofectamine™ 2000 were mixed with 3400 μL (3.4 mL) of DMEMmedium and incubated for 5 minutes at RT.

Preparation of Test and Control Molecule Working Solutions:

working solutions at various concentrations was prepared by diluting a10 μM stock solution. This dilution series was prepared for thegeneration of final transfection concentrations ranging between 0.0095nM and 100 nM in 100 μL DMEM transfection medium (0.0095, 0.019, 0.039,0.07, 0.15, 0.31, 0.625, 1.25, 2.5, 5.0, 20.0, 100.0).

100 μL aliquots of the diluted Lipofectamine 2000 were mixed gently with100 μL of each of the diluted test molecule or control molecule workingsolutions (above) and mixtures were incubated for 20-40 minutes at RT.Following incubation, 20 μL of the siRNA/Lipofectamine™ 2000mixture wereadded on top of the cells (pre-incubated with 80 μL of cell-culturemedium above). The plates were gently rocked. Cells were incubated for48 hours at 37±1° C. and 5% CO₂.

Determination of Renilla Luciferase Activity in Transfected Cells

The psiCHECK™-2 vector enables monitoring of changes in expression of atarget sequence fused to the Renilla luciferase reporter gene. Thetest/control molecule target sequence is cloned into the 3′-untranslatedregion (3′-UTR) of Renilla luciferase. Measuring the decrease in Renillaluciferase activity thus provides a convenient way of monitoringactivity. In addition, the psiCHECK™-2 vector contains a second reportergene, Firefly luciferase, transcribed under a different promoter, whichallows for normalization of Renilla luciferase expression.

48 Hours following test or control molecule, transfection Renilla andFireFly Luciferase activities were measured in each of the siRNAtransfected samples, using Dual-Luciferase® Assay kit (Promega)according to manufacturer procedure:

Medium was completely removed from cells and cells were then lysed bythe addition of 40 μL/well 1× Luciferase lysis solution. Plates werethen frozen (−80° C.) and thawed at RT. Cell lysates were suspended bypipetting several times and aliquots of 12.5 μL of each sample weretransferred to a separate 96-well plate. 50 μL Luciferase substrate(LARII) was added to each extract and Firefly Luciferase activity wasmeasured by Absorbance, Fluorescence and Luminescence Reader (PerkinElmer, Victor™ 1240). 50 μL of Stop&Glo Reagent was added to each of thesamples and Renilla Luciferase activity was measured immediately.Renilla Luciferase activity value was divided by Firefly Luciferaseactivity value for each sample (normalization) Renilla luciferaseactivity is finally expressed as the percentage of the normalizedactivity value in tested sample relative to the normalized valueobtained in cells transfected with the corresponding psiCHECK™-2 plasmidin the absence of test or control molecules.

IC50 Calculation

The IC50 values of test and control molecule activity against the GS_CMsite were determined by constructing a dose-response curve using theactivity results obtained with the various final siRNA concentrations.The dose response curve was constructed by plotting the relativenormalized values of Renilla luciferase activity versus the logarithm oftransfected siRNA concentration. The curve was calculated by fitting thebest sigmoid curve to the measured data. The methods for the sigmoid fitis called 3-point curve fit.

$Y = {{Bot} + \frac{100 - {Bot}}{1 + 10^{{({{{LogIC}\; 50} - X})} \times {HillSlope}}}}$Where:Y is the residual caspase 2 mRNA response,X is the logarithm of transfected siRNA concentration,Bot is the Y value at the bottom plateau,LogIC50 is the X value when Y is halfway between bottom and top plateausand HillSlope is the steepness of the curve.Results

For the evaluation of the potential off-target activity of each strandof a double stranded RNA molecule strands, the“guide-seed-sequence-and-passenger-strand-based activity assay” wasemployed using the psiCHECK (Promega) plasmid constructs. Themeasurement of Renilla activity provides a convenient way of monitoringdouble stranded RNA activity.

Measurement of Target Renilla Luciferase Protein Activity

The activity of both test and control molecules against the four targetsequences (GS-CM, guide strand complete match; GS-SM, guide strand seedmatch; PS-CM, passenger strand complete match; and PS-SM, passengerstrand seed match) was assessed at the protein level by measuring therelative activity of the Renilla luciferase reporter protein in cellstransfected with various concentrations of molecule. Each assay wasrepeated three times. The IC50 value of test molecule activity againstthe GS-CM target site was determined by construction ofconcentration-response plots.

Both test and control molecules were active against the target GS-CMsite in a dose response.

Both siRNAs were inactive against the GS-SM and the PS-SM sites.

FIGS. 2-10 and Tables 2-10 below show activity as measured by residualtarget for double stranded molecules targeting the complete match of atarget sequence which has a substitution at position 19 (equivalent ofposition 1 of the antisense) to incorporate one of the ribonucleotides,adenosine (A or rA), cytidine (C or rC), guanosine (G or rG),ribothymidine (rT, also 5′ methyluridine) and uridine (U or rU); ordeoxyadenosine (dA), deoxycytidine (dC), deoxyguanosine (dG), thymidine(dT) and deoxyuridine (dU); or 2′O methylated adenosine (mA), 2′Omethylated cytidine (mC), 2′O methylated guanosine (mG), 2′O methylateduridine (mU). “1st pos AS” refers to position 1 of the antisense strand(5′ terminal nucleotide).

TABLE 2 TLR2_16 siRNA (blunt ended with alternating 2′-OMe on bothstrands) 1st pos 1st pos 1st pos 1st pos AS -mC AS -mG AS - mA AS - dUsiRNA 0.4 nM Target G 81 75 68 58 concen- Target U 91 93 81 59 tration 4nM Target G 85 61 50 38 Target U 84 78 56 40 20 nM Target G 57 55 38 32Target U 62 62 35 32

The data shown in Table 2 is presented in FIG. 2. Methylated nucleotidesat position 1 of the antisense strand show reduced target knockdown,irrespective of base pairing.

TABLE 3 TLR2_37 siRNA unmodified with C3-C3 overhangs or dTdT overhangsdTdT overhang C3-C3 overhang Position 1st pos 1st pos 1st pos 1st pos1st pos 1st pos 19 target AS -U AS -U AS - dU AS - dT AS - mU AS - AsiRNA 0.04 nM Target A 21 18 17 16 48 18 concen- Target C 20 15 22 21 396 tration Target G 35 30 27 27 60 24 Target U 33 33 28 32 64 27 0.4 nMTarget A 13 8 8 9 23 11 Target C 12 9 9 11 18 10 Target G 17 13 12 13 3416 Target U 19 17 12 16 43 14 4 nM Target A 10 8 7 7 13 9 Target C 8 7 89 12 10 Target G 13 9 8 11 20 10 Target U 12 13 8 9 22 12

The data in Table 3 is presented in FIG. 3. Table 3 and FIG. 3 show thata 2′OMe ribonucleotide in the 1st position of AS reduces siRNA activityregardless of the nucleotide in the matching position of the target.

TABLE 4 TLR2_37 siRNA unmodified with C3-C3 overhangs or dTdT overhangsdTdT overhang C3-C3 overhang 1st pos 1st pos 1st pos 1st pos AS -U AS -UAS - dU AS - dT siRNA 0.04 nM Target A 21 18 17 16 concen- Target C 2015 22 21 tration Target G 35 30 27 27 Target U 33 33 28 32 0.4 nM TargetA 13 8 8 9 Target C 12 9 9 11 Target G 17 13 12 13 Target U 19 17 12 16

The data in Table 4 is presented in FIG. 4. Table 4 and FIG. 4 show that

1. siRNA with highest activity is observed when U (dU or dT) in the 1stposition of AS is mismatched to C in the target or is matched to A inthe target.

2. If U in the 1st position of AS of siRNA (or dU or dT) is mismatchedto U in the target, siRNA activity is reduced

3. If U in the 1st position of AS is wobble-paired with G in target,siRNA activity is reduced.

TABLE 5 TLR2_37 siRNA (non-modified with dTdT overhangs) - 0.04 nM 1stpos AS - G 1st pos AS - U SiRNA 0.04 nM   Target A 32 21 concentrationTarget C 29 20 Target G 33 35 Target U 37 33 0.4 nM  Target A 14 13Target C 13 12 Target G 17 17 Target U 21 19  4 nM Target A 10 10 TargetC 13 8 Target G 10 13 Target U 11 12 20 nM Target A 9 11 Target C 8 8Target G 8 11 Target U 10 11 100 nM  Target A 8 7 Target C 5 7 Target G7 8 Target U 5 7

The data is Table 5 is presented in FIG. 5. Table 5 and FIG. 5 show that

1. U in the 1st position of the AS produced better results with itsmatched A or mismatched C in the target than with mismatched U orwobble-paired G.

2. G in the 1st position of the AS showed similar results with allpossible nucleotides in the matching target position—mismatches orwobble-pairing did not improve activity.

TABLE 6 TLR2_46 siRNA (unmodified with C3-C3 or dTdT overhangs) dTdToverhang C3-C3 overhang 1st pos 1st pos 1st pos 1st pos 1st pos 1st posAS -U AS -U AS - dU AS - dT AS - mU AS - A siRNA 0.04 nM Target A 70 8361 69 79 52 concen- Target C 56 78 49 66 85 58 tration Target U 68 85 5769 95 67 0.4 nM Target A 41 30 33 36 83 37 Target C 35 39 33 41 86 34Target U 56 46 43 36 94 48 4 nM Target A 13 16 17 20 39 14 Target C 1517 11 24 48 13 Target U 19 21 17 14 59 19 20 nM Target A 8 7 8 8 28 10Target C 7 10 7 8 27 9 Target U 10 12 10 6 40 13

The data in Table 6 is presented in FIG. 6. Table 6 and FIG. 6 show thatsubstitution of U with mU reduces activity

TABLE 7 TLR2_46 siRNA (non-modified with dTdT overhangs) 1st pos AS - G1st pos AS - U siRNA 0.4 nM  Target A 53 41 concentration Target C 61 35Target U 68 56  4 nM Target A 28 13 Target C 28 15 Target U 32 19 20 nMTarget A 16 8 Target C 15 7 Target U 17 10 100 nM  Target A 16 8 TargetC 15 7 Target U 17 10

The data in Table 7 is presented in FIG. 7. Table 7 and FIG. 7 show thatregardless of the nucleotide type of the matching target position, U inthe 1st position of AS produced better KD (knock-down). All casesmean—regardless of the complementarity between siRNA and target in the1st position of the guide strand, or mismatch or wobble pair.

TABLE 8 RHOA_61 siRNA (non-modified/C3-C3 or dTdT overhangs) dTdToverhang C3-C3 overhang 1st pos 1st pos 1st pos 1st pos 1st pos 1st posAS -U AS -U AS - dU AS - dT AS - mU AS - A siRNA 0.04 nM Target A 35 5034 33 76 33 concen- Target C 32 38 33 32 86 34 tration Target G 41 51 49Target U 53 34 36 0.4 nM Target A 26 24 26 25 74 24 Target C 26 22 22 6623 Target G 26 26 38 35 Target U 36 27 27 4 nM Target A 22 22 24 17 3720 Target C 18 19 21 17 34 18 Target G 21 31 25 Target U 30 22 22

The data in Table 8 is presented in FIG. 8.

TABLE 9 RHOA_61 siRNA (non-modified/C3-C3 or dTdT overhangs) dTdToverhang C3-C3 overhang 1st pos 1st pos 1st pos 1st pos 1st pos 1st posAS -U AS -U AS - dU AS - dT AS - mU AS - A siRNA 0.04 nM Target A 35 5034 33 76 33 concen- Target C 32 38 33 32 86 34 tration Target G 41 51 49Target U 53 34 36 0.4 nM Target A 26 24 26 25 74 24 Target C 26 22 22 6623 Target G 26 26 38 35 Target U 36 27 27 4 nM Target A 22 22 24 17 3720 Target C 18 19 21 17 34 18 Target G 21 31 25 Target U 30 22 22

The data in Table 9 is presented in FIG. 9. Table 9 and FIG. 9 show thatsiRNAs with U in the 1st position of the AS are more active than thosewith G in the 1st position regardless of the nucleotide in the matchingposition of the target (match, mismatch, wobble). The results cannot beexplained by Wobble base-pairing since having G in siRNA and U in thetarget is not equivalent to having U in siRNA and G in the target

TABLE 10 RhoA_61 siRNA (unmodified with dTdT overhangs) 1st pos AS - G1st pos AS - U siRNA 0.04 nM   Target A 52 35 concentration Target C 5232 Target G 90 Target U 66 0.4 nM  Target A 49 26 Target C 51 26 TargetG 84 Target U 49  4 nM Target A 23 22 Target C 22 18 Target G 38 TargetU 28 20 nM Target A 21 20 Target C 18 17 Target G 29 Target U 21 100 nM Target A 18 19 Target C 19 16 Target G 25 Target U 20

The data in Table 10 is presented in FIG. 10.

Example 3 Model Systems of Acute Renal Failure (ARF)

ARF is a clinical syndrome characterized by rapid deterioration of renalfunction that occurs within days. Without being bound by theory theacute kidney injury may be the result of renal ischemia-reperfusioninjury such as renal ischemia-reperfusion injury in patients undergoingmajor surgery such as major cardiac surgery. The principal feature ofARF is an abrupt decline in glomerular filtration rate (GFR), resultingin the retention of nitrogenous wastes (urea, creatinine) Recentstudies, support that apoptosis in renal tissues is prominent in mosthuman cases of ARF. The principal site of apoptotic cell death is thedistal nephron. During the initial phase of ischemic injury, loss ofintegrity of the actin cytoskeleton leads to flattening of theepithelium, with loss of the brush border, loss of focal cell contacts,and subsequent disengagement of the cell from the underlying substratum.

The compounds of the invention are tested for efficacy in treatingischemia reperfusion injury in an animal model ofischemia-reperfusion-induced ARF.

Example 4 Model Systems of Pressure Sores or Pressure Ulcers

Pressure sores or pressure ulcers including diabetic ulcers, are areasof damaged skin and tissue that develop when sustained pressure (usuallyfrom a bed or wheelchair) cuts off circulation to vulnerable parts ofthe body, especially the skin on the buttocks, hips and heels. The lackof adequate blood flow leads to ischemic necrosis and ulceration of theaffected tissue. Pressure sores occur most often in patients withdiminished or absent sensation or who are debilitated, emaciated,paralyzed, or long bedridden. Tissues over the sacrum, ischia, greatertrochanters, external malleol, and heels are especially susceptible;other sites may be involved depending on the patient's situation.

The compounds of the invention are tested for efficacy in treatingpressure sores, ulcers and similar wounds in, inter alia, the mousemodel as described in Reid et al., J. Surg. Res. 116:172-180, 2004 or inthe rabbit model as described by Mustoe et al, JCI, 1991. 87(2):694-703;Ahn and Mustoe, Ann P1 Surg, 1991. 24(1):17-23.

Example 5 Model Systems of Chronic Obstructive Pulmonary Disease (COPD)

Chronic obstructive pulmonary disease (COPD) is characterized mainly byemphysema, which is permanent destruction of peripheral air spaces,distal to terminal bronchioles. Emphysema is also characterized byaccumulation of inflammatory cells such as macrophages and neutrophilsin bronchioles and alveolar structures. Emphysema and chronic bronchitismay occur as part of COPD or independently.

The compounds of the invention are tested for efficacy in treatingCOPD/emphysema/chronic bronchitis in, inter alia, animal models such asthose disclosed as follows:

Starcher and Williams, 1989. Lab. Animals, 23:234-240; Peng, et al.,2004.; Am J Respir Crit Care Med, 169:1245-1251; Jeyaseelan et al.,2004. Infect. Immunol, 72: 7247-56. Additional models are described inPCT patent publication WO 2006/023544 assigned to the assignee of thepresent application, which is hereby incorporated by reference into thisapplication.

Example 6 Model Systems of Spinal Cord Injury

Spinal cord injury, or myelopathy, is a disturbance of the spinal cordthat results in loss of sensation and/or mobility. The two common typesof spinal cord injury are due to trauma and disease. Traumatic injurycan be due to automobile accidents, falls, gunshot, diving accidentsinter alia, and diseases which can affect the spinal cord include polio,spina bifida, tumors and Friedreich's ataxia.

siRNA is injected into the spinal cord following spinal cord contusionand in uninjured rats. Sagittal cryosections are produced andimmunostaining using four different groups of antibodies is performed inorder to determine whether uptake has occurred in neurons, astroglia,oligdendroglia and/or macrophages/microglia. Markers for neurons includeNeuN, or GAP43; markers for astroglia and potential neural stem cellsinclude GFAP, nestin or vimentin; markers for oligdendroglia include NG2or APC; markers for macrophages/microglia include ED1 or Iba-1 (Hasegawaet al., 2005. Exp Neurol 193:394-410).

Rats are injected with two different doses of siRNA (1 μg/ul, 10 μg/μl)and are left for 1 and 3 days before sacrifice. The results indicatethat siRNA to spinal cord injury target genes increases motoneuronrecovery.

Example 7 Model Systems of Glaucoma

The compounds of the invention are tested for efficacy in treating orpreventing glaucoma in the animal model, for example, as described byPease et al., J. Glaucoma, 2006, 15(6):512-9 (Manometric calibration andcomparison of TonoLab and TonoPen tonometers in rats with experimentalglaucoma and in normal mice).

Example 7A Model Systems of Ischemic Optic Neuropathy (ION)

An animal model for Ischemic optic neuropathy was established in adultsWistar rats using a protocol of optic nerve crush injury. Seven daysprior to the optic nerve crush, the retinal ganglion cells (RGC) areselectively labelled by application of the retrograde tracer FluoroGold(2%, Fluorochrome, Englewood, Colo.) to the superior colliculus. Thetracer is transported by retrograde transport along RGC axons resultingin complete and specific labelling of all RGCs within 1 week postinjection of the fluorescent tracer. The animals are subjected to theoptic nerve crush injury 7 days post retrograde tracing. The orbitaloptic nerve is exposed through a supraorbital approach and all axons inthe optic nerve were transected by crushing with forceps for 10 seconds,2 mm from the lamina cribrosa. A single dose of 20 μg/5 μl of PBS of thetest modified siRNA compound is microinjected into the vitreous body 2mm anterior to the nerve head, using a glass micropipette at the time ofthe optic nerve crush.

The survival of RGCs is determined 7 days following the optic nervecrush by counting FluoroGold-labelled RGCs on flat-mounted retinas. Theexperimental animals are perfused transcardially with 4%paraformaldehyde at 1 week after the optic nerve crush. Both retinas aredissected out, fixed for an additional 30 min and flat-mounted on aglass slide for ganglion cell layer quantification. The number offluorescent RGCs is counted in 16 distinct areas in each retina and thepercent of survival of the RGCs is determined compared to samplesobtained from rats which did not undergo optic nerve crush injury at allor samples obtained from rats which were injected with PBS, controlsiRNA or GFP siRNA along with the optic nerve crush injury. Microgliacells that may have incorporated FluoroGold after phagocytosis of dyingRGCs were distinguished by their characteristic morphology and excludedfrom quantitative analyses.

Another model of optic nerve axotomy where the entire population of RGCsare axotomized by transecting the optic nerve close to the eye isutilized. (Cheng L, et al. J. Neurosci. 2002; 22:3977-3986).

Example 8 Model Systems of Ischemia/Reperfusion Injury Following LungTransplantation in Rats

The compounds of the invention are tested for efficacy in treatingischemia/reperfusion injury or hypoxic injury following lungtransplantation in one or more of the experimental animal models, forexample as described by Mizobuchi et al., 2004. J. Heart LungTransplant, 23:889-93; Huang, et al., 1995. J. Heart Lung Transplant.14: S49; Matsumura, et al., 1995. Transplantation 59: 1509-1517; Wilkes,et al., 1999. Transplantation 67:890-896; Naka, et al., 1996.Circulation Research, 79: 773-783.

Example 9 Model Systems of Acute Respiratory Distress Syndrome

The compounds of the invention are tested for efficacy in treating acuterespiratory distress syndrome in inter alia in the animal modeldescribed by Chen et al (J Biomed Sci. 2003; 10(6 Pt 1):588-92. ModifiedsiRNA compounds that target genes including CYBA, HRK, BNIP3, MAPK8,MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, SPP1, and DUOX1 are tested inthis animal model.

Example 10 Model Systems of Hearing Loss Conditions

(i) Chinchilla Model of Carboplatin-Induced or Cisplatin-Induced CochleaHair Cell Death

Chinchillas are pre-treated by direct administration of specific siRNAin saline to the left ear of each animal. Saline is given to the rightear of each animal as placebo. Two days following the administration ofthe specific modified siRNA compounds of the invention, the animals aretreated with carboplatin (75 mg/kg ip) or cisplatin (intraperitonealinfusion of 13 mg/kg over 30 minutes). After sacrifice of thechinchillas (two weeks post carboplatin treatment) the % of dead cellsof inner hair cells (IHC) and outer hair cells (OHC) is calculated inthe left ear (siRNA treated) and in the right ear (saline treated). Itis calculated that the % of dead cells of inner hair cells (IHC) andouter hair cells (OHC) is lower in the left ear (siRNA treated) than inthe right ear (saline treated).

(ii) Chinchilla Model of Acoustic-Induced Cochlea Hair Cell Death

The activity of specific siRNA in an acoustic trauma model is studied inchinchilla. The animals are exposed to an octave band of noise centeredat 4 kHz for 2.5 h at 105 dB. The left ear of the noise-exposedchinchillas is pre-treated (48 h before the acoustic trauma) with 30 μgof siRNA in ˜10 μL of saline; the right ear is pre-treated with vehicle(saline). The compound action potential (CAP) is a convenient andreliable electrophysiological method for measuring the neural activitytransmitted from the cochlea. The CAP is recorded by placing anelectrode near the base of the cochlea in order to detect the localfield potential that is generated when a sound stimulus, such as clickor tone burst, is abruptly turned on. The functional status of each earis assessed 2.5 weeks after the acoustic trauma. Specifically, the meanthreshold of the compound action potential recorded from the roundwindow is determined 2.5 weeks after the acoustic trauma in order todetermine if the thresholds in the siRNA-treated ear are lower (better)than the untreated (saline) ear. In addition, the level of inner andouter hair cell loss is determined in the siRNA-treated and the controlear.

Similar models are used in mice and rats. The modified siRNA compoundsthat target genes including BNIP3, CAPNS, HEST, HESS, NOX3, ID1-3, HRK,ASPP2 (TP53BP), CASP2, RAC1, HTRA2, CDKN1B are tested in these and otheranimal model of hearing loss and hearing regeneration.

Example 11 Animal Models of Osteoarthritis (OA)

Collagen induced arthritis (CIA): CIA in mice is described in Trenthamet al. (1977. J. Exp. Med. 146: 857-868). Adjuvant-induced arthritis(AA):AA is described in Kong et al., (1999. Nature, 402:304-308). Amenisectomy model is described in Han et al., (1999. Nagoya J Med Sci62(3-4):115-26).

The effect of different siRNA inhibitors, such as siRNA to SSPi, ondifferent parameters related to OA such as chondrocyte proliferation,terminal differentiation and development of arthritis, is evaluatedusing one or more of the above models, in addition to in vitro modelsknown in the art. Modified siRNA compounds directed to proapoptoticgenes, in particular to SSP1, are tested in these animal models whichshow that the modified siRNA compounds treat and/or prevent OA and thusmay be used to treat this condition.

Example 12 Rat Model Systems for Transplantation-Associated Acute KidneyInjury

Warm Ischemia—

In test rats a left nephrectomy is performed, followed by autotransplantation that results in a warm kidney graft preservation periodof 45 minutes. Following auto transplantation, a right nephrectomy isperformed on the same animal. Chemically modified siRNA to a target isadministered intravenously via the femoral vein either before harvestingof the kidney graft (mimicking donor treatment) (“pre”), or after thekidney autotransplantation (mimicking recipient treatment), or bothbefore harvest and after transplantation (combined donor and recipienttreatment) (“pre-post”).

Cold Ischemia—

A left nephrectomy is performed on a donor animal, followed by a coldpreservation (on ice) of the harvested kidney for a period of 5 hours.At the end of this period, the recipient rat will undergo a bilateralnephrectomy, followed by transplantation of the cold-preserved kidneygraft. The total warm ischemia time (including surgical procedure) isabout 30 minutes. Chemically modified siRNA is administeredintravenously via the femoral vein, either to the donor animal prior tothe kidney harvest (“pre”), or to the recipient animal 15 minutes (“post15 min”) or 4 hours (post 4 hrs) post-transplantation.

To assess the efficacy of the modified siRNA compounds of the presentinvention in improving post-transplantation renal function, serumcreatinine levels are measured on days 1, 2, and 7 post-transplantationin both warm and cold ischemia models.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The invention claimed is:
 1. A double-stranded nucleic acid havingstructure (A) set forth below: (A) 5′    N¹-(N)x-Z 3′ (antisense strand)3′ Z′-N²-(N′)y-z″ 5′ (sense strand)

wherein each of N², N and N′ is an unmodified ribonucleotide, a modifiedribonucleotide, or an unconventional moiety; wherein each of (N)x and(N′)y is an oligonucleotide in which each consecutive N or N′ is joinedto the adjacent N or N′ by a covalent bond; wherein x=y=18; wherein thesequence of N²—(N′)y is fully complementary to the sequence of N¹—(N)xand the sequence of (N)x has complementarity to a consecutive sequencein a target RNA; wherein N¹ is covalently bound to (N)x and notcomplementary to the corresponding nucleotide in the target RNA or is anunconventional moiety complementary to the target RNA; wherein: N¹ isselected from the group consisting of adenosine and deoxyadenosine whenthe corresponding nucleotide in the target RNA sequence is adenosine; orN¹ is selected from the group consisting of adenosine, deoxyadenosineand deoxythymidine when the corresponding nucleotide in the target RNAsequence is cytidine; or N¹ is selected from the group consisting ofadenosine, deoxyadenosine and deoxythymidine when the correspondingnucleotide in the target RNA sequence is guanosine; or N¹ is selectedfrom the group consisting of uridine and deoxyuridine when thecorresponding nucleotide in the target RNA sequence is uridine; whereinz″ may be present or absent, but if present is a capping moietycovalently attached at the 5′ terminus of N²—(N′)y; and wherein each ofZ and Z′ is independently present or absent, but if present isindependently 1-5 consecutive nucleotides, 1-5 consecutivenon-nucleotide moieties or a combination of 1-5 nucleotides andnon-nucleotide moieties, covalently attached at the 3′ terminus of thestrand in which it is present; or a pharmaceutically acceptable salt ofthe double stranded nucleic acid.
 2. A double-stranded nucleic acidhaving structure (A) set forth below: (A) 5′    N¹-(N)x-Z 3′(antisense strand) 3′ Z′-N²-(N′)y-z″ 5′ (sense strand)

wherein each of N², N and N′ is an unmodified ribonucleotide, a modifiedribonucleotide, or an unconventional moiety; wherein each of (N)x and(N′)y is an oligonucleotide in which each consecutive N or N′ is joinedto the adjacent N or N′ by a covalent bond; wherein x=y=18; wherein thesequence of N²—(N′)y is fully complementary to the sequence of N¹—(N)xand the sequence of (N)x has complementarity to a consecutive sequencein a target RNA; wherein N¹ is covalently bound to (N)x and notcomplementary to the corresponding nucleotide in the target RNA or is anunconventional moiety complementary to the target RNA; wherein N¹ isselected from the group consisting of adenosine and deoxyadenosine, andthe corresponding nucleotide in the target RNA is adenosine; wherein z″may be present or absent, but if present is a capping moiety covalentlyattached at the 5′ terminus of N²—(N′)y; and wherein each of Z and Z′ isindependently present or absent, but if present is independently 1-5consecutive nucleotides, 1-5 consecutive non-nucleotide moieties or acombination of 1-5 nucleotides and non-nucleotide moieties, covalentlyattached at the 3′ terminus of the strand in which it is present; or apharmaceutically acceptable salt of the double stranded nucleic acid. 3.The double-stranded nucleic acid of claim 2, wherein N1 is adenosine. 4.The double-stranded nucleic acid of claim 1 wherein z″ is present. 5.The double-stranded nucleic acid of claim 1, wherein Z and Z′ areabsent.
 6. The double-stranded nucleic acid of claim 1, wherein one of Zor Z′ is present.
 7. The double-stranded nucleic acid of claim 6,wherein Z or Z′ comprise non-nucleotide moieties.
 8. The double-strandednucleic acid of claim 1, wherein at least one of N or N′ comprises a2′O-methyl sugar modified ribonucleotide.
 9. The double-stranded nucleicacid of claim 8, wherein (N)x comprises 2′O-methyl sugar modifiedribonucleotides.
 10. The double-stranded nucleic acid of claim 1,wherein the double-stranded nucleic acid is a siRNA, siNA or a miRNA.11. A double-stranded nucleic acid having structure (A) set forth below:(A) 5′    N¹-(N)x-Z 3′ (antisense strand) 3′ Z′-N²-(N′)y-z″ 5′(sense strand)

wherein N, N′, (N)x, (N′)y, x, y, Z′ and z″ are defined in claim 2;wherein the sequence of N²—(N′)y is fully complementary to the sequenceof N¹—(N)x and the sequence of (N)x has complementarity to consecutivesequence in a target RNA; wherein N¹ is covalently bound to (N)x and notcomplementary to the corresponding nucleotide in the target RNA or is anunconventional moiety complementary to the target RNA; wherein, Z′ isabsent or present, Z is present and comprises two alkyl moietiescovalently linked to each other via a phosphodiester bond, wherein N²comprises uridine or deoxyuridine; and wherein N¹ comprises adenosinewhen the corresponding nucleotide in the target RNA is adenosine,guanosine or cytidine; or a pharmaceutically acceptable salt the doublestranded nuclei acid.
 12. The double-stranded nucleic acid of claim 1,wherein the target RNA is an mRNA encoded by a mammalian gene.
 13. Thedouble-stranded nucleic acid of claim 12, wherein the target mRNA is anmRNA encoded by a human gene.
 14. A composition comprising at least onedouble-stranded nucleic acid or a pharmaceutically acceptable salt ofsuch double-stranded nucleic acid of claim 1; and a pharmaceuticallyacceptable carrier.
 15. A method for treating or preventing theincidence or severity of a disease or condition in a subject in needthereof, comprising administering to the subject a double-strandednucleic acid of claim 1 in an amount effective to prevent or treat ordelay the onset of the disease or the condition, wherein the disease orthe condition and/or symptoms associated therewith is selected from thegroup consisting of hearing loss, acute renal failure (ARF), DelayedGraft Function (DGF) after kidney transplantation, glaucoma, ocularischemic conditions, including non-arteric ischemic optic neuropathy(NAION), anterior ischemic optic neuropathy, age-related maculardegeneration (AMD), Ischemic Optic Neuropathy (ION) and dry eyesyndrome, acute respiratory distress syndrome (ARDS) and other acutelung and respiratory injuries, chronic obstructive pulmonary disease(COPD), primary graft failure, ischemia-reperfusion injury, reperfusioninjury, reperfusion edema, allograft dysfunction, pulmonaryreimplantation response and/or primary graft dysfunction (PGD) afterorgan transplantation, in particular in lung transplantation, organtransplantation including lung, liver, heart, pancreas, and kidneytransplantation, nephro- and neurotoxicity, spinal cord injury, braininjury, neurodegenerative disease or condition, pressure sores, oralmucositis, fibrotic disorders and cancer.
 16. A double-stranded nucleicacid molecule having structure (A) set forth below: (A) 5′    N¹-(N)x-Z3′ (antisense strand) 3′ Z′-N²-(N′)y-z″ 5′ (sense strand)

wherein N², N, N′, (N)x, (N′)y, x, y, Z, Z′ and z″ are as defined inclaim 2; wherein the sequence of N²—(N′)y is fully complementary to thesequence of N¹—(N)x and the sequence of (N)x has complementarity toconsecutive sequence in a target RNA; wherein N¹ is covalently bound to(N)x and not complementary to the corresponding nucleotide in the targetRNA or is an unconventional moiety complementary to the target RNA;wherein N¹ is selected from the group consisting of adenosine,deoxyadenosine and deoxythymidine, when the corresponding nucleotide inthe target RNA is cytidine; or a pharmaceutically acceptable salt of thedouble stranded nucleic acid.
 17. A double-stranded nucleic acidmolecule having structure (A) set forth below: (A) 5′    N¹-(N)x-Z 3′(antisense strand) 3′ Z′-N²-(N′)y-z″ 5′ (sense strand)

wherein N², N, N′, (N)x, (N′)y, x, y, Z, Z′ and z″ are as defined inclaim 2; wherein the sequence of N²—(N′)y is fully complementary to thesequence of N¹—(N)x and the sequence of (N)x has complementarity to aconsecutive sequence in a target RNA; wherein N¹ is covalently bound(N)x and not complementary to the corresponding nucleotide in the targetRNA or is an unconventional moiety complementary to the target RNAwherein N¹ is selected from the group consisting of adenosine,deoxyadenosine and deoxythymidine when the corresponding nucleotide inthe target RNA is guanosine; or a pharmaceutically acceptable salt ofthe double stranded nucleic acid.
 18. A double-stranded nucleic acidmolecule having structure (A) set forth below: (A) 5′    N¹-(N)x-Z 3′(antisense strand) 3′ Z′-N²-(N′)y-z″ 5′ (sense strand)

wherein N², N, N′, (N)x, (N′) y, x, y, Z, Z′ and z″ are as defined inclaim 2; wherein the sequence of N²—(N′)y is fully complementary to thesequence of N¹—(N)x and the sequence of (N)x has complementarity toconsecutive sequence in a target RNA; wherein N¹ is covalently bound to(N)x and not complementary to the corresponding nucleotide in the targetRNA or is an unconventional moiety complementary to the target RNA;wherein N¹ is uridine or deoxyuridine, when the corresponding nucleotidein the target RNA is uridine; or a pharmaceutically acceptable salt ofthe double stranded nucleic acid.